Nucleic acid molecules encoding alanine 2,3-aminomutases

ABSTRACT

Alanine 2,3-aminomutase sequences are disclosed, as are cells having alanine 2,3-aminomutase activity and methods of selecting for such cells. Methods for producing beta-alanine, pantothenate, 3-hydroxypropionic acid, as well as other organic compounds, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 11/938,154filed Nov. 9, 2007, now U.S. Pat. No. 7,655,451, which is a divisionalof U.S. patent application Ser. No. 10/502,040, filed Jul. 19, 2004, nowU.S. Pat. No. 7,309,597, which is the U.S. National Stage ofInternational Application No. PCT/US03/01635, filed Jan. 17, 2003(published in English under PCT Article 21(2)), which in turn claims thebenefit of U.S. Patent Applications 60/350,727 filed Jan. 18, 2002 and60/375,785 filed Apr. 25, 2002.

FIELD

This disclosure relates to alanine 2,3-aminomutase nucleic acid andamino acid sequences, cells having alanine 2,3-aminomutase activitywhich can convert alpha-alanine to beta-alanine, and methods using thesecells to make beta-alanine, pantothenic acid, 3-hydroxypropionic acid,and other organic compounds.

BACKGROUND

Organic chemicals such as organic acids, esters, and polyols can be usedto synthesize plastic materials and other products. To meet theincreasing demand for organic chemicals, more efficient andcost-effective production methods are being developed which utilize rawmaterials based on carbohydrates rather than hydrocarbons. For example,certain bacteria have been used to produce large quantities of lacticacid used in the production of polylactic acid.

3-hydroxypropionic acid (3-HP) is an organic acid. Several chemicalsynthesis routes have been described to produce 3-HP, and biocatalyticroutes have also been disclosed (WO 01/16346 to Suthers et al.). 3-HPhas utility for specialty synthesis and can be converted to commerciallyimportant intermediates by known art in the chemical industry, e.g.,acrylic acid by dehydration, malonic acid by oxidation, esters byesterification reactions with alcohols, and 1,3-propanediol byreduction.

SUMMARY

The compound 3-hydroxypropionic acid (3-HP) can be producedbiocatalyticly from PEP or pyruvate, through a key beta-alanineintermediate (FIG. 1). Beta-alanine can be synthesized in cells fromcarnosine, beta-alanyl arginine, beta-alanyl lysine, uracil via5,6-dihydrouracil and N-carbamoyl-beta-alanine, N-acetyl-beta-alanine,anserine, or aspartate (FIGS. 1 and 2). However, these routes arerelatively inefficient because they require rare precursors or startingcompounds that are more valuable than 3-HP.

Therefore, production of 3-HP using biocatalytic routes would be moreefficient if alpha-alanine could be converted to beta-alanine directly(FIG. 1). Unfortunately, an enzyme that interconverts alpha-alanine tobeta-alanine has not yet been identified. It would be advantageous ifenzymatic activities that carry out the conversion of alpha-alanine tobeta-alanine were identified, such as an alanine 2,3-aminomutase.

Herein disclosed are alanine 2,3-aminomutase nucleic acid sequences(such as SEQ ID NOS: 20 and 29), amino acid sequences (such as SEQ IDNOS: 21 and 30), as well as variants, fragments, fusions, andpolymorphisms thereof that retain alanine 2,3-aminomutase activity. Inone example, the polypeptide is a sequence that includes SEQ ID NO: 21or 30, or variants, fragments, or fusions thereof that retain alanine2,3-aminomutase activity. In one example, the polypeptide is a mutatedlysine 2,3-aminomutase and/or a lysine 5,6-aminomutase amino acidsequence. The disclosed sequences can be used to transform cells, suchthat the transformed cells have alanine 2,3-aminomutase activity, whichallows the cells to produce beta-alanine from alpha-alanine. Bindingagents that specifically bind to an alanine 2,3-aminomutase areencompassed by this disclosure.

Cells having alanine 2,3-aminomutase activity, which allow the cell toconvert alpha-alanine to beta-alanine, are disclosed. Such cells can beeukaryotic or prokaryotic cells, such as yeast cells, plant cells,Lactobacillus, Lactococcus, Bacillus, or Escherichia cells. In oneexample, the cell is transformed with a mutated lysine 2,3-aminomutaseand/or a mutated lysine 5,6-aminomutase that confers to the transformedcells alanine 2,3-aminomutase activity. In another example, transformedcells include an alanine 2,3-aminomutase, such as SEQ ID NO: 21 or 30.The disclosed cells can be used to produce nucleic acid molecules,polypeptides, and organic compounds. The polypeptides can be used tocatalyze the formation of organic compounds or can be used as antigensto create specific binding agents.

A production cell having at least one exogenous nucleic acid, such as anucleic acid encoding for an alanine 2,3-aminomutase, is disclosed. Inone example, the nucleic acid sequence includes SEQ ID NOS: 20 or 29 (orfragments, variants, or fusions thereof that retain alanine2,3-aminomutase activity). In another example, the nucleic acid sequenceencodes an amino acid sequence shown in SEQ ID NO: 21 or 30 (orfragments, variants or fusion proteins that of that retain alanine2,3-aminomutase activity). Production cells can be used to expresspolypeptides that have an enzymatic activity such as CoA transferaseactivity, beta-alanine ammonia lyase activity, 3-hydroxypropionyl-CoA(3-HP—CoA) dehydratase activity, glutamate dehydrogenase,3-hydroxypropionyl-CoA hydrolase, alanine dehydrogenase,pyruvate-glutamate transaminase, and/or 3-hydroxyisobutyryl-CoAhydrolase activity. In another example, production cells are used toexpress polypeptides that have an enzymatic activity such asbeta-alanine-2-oxoglutarate aminotransferase and 3-HP dehydrogenaseand/or 3-hydroxyisbutyrate dehydrogenase. Methods of producingpolypeptides encoded by the nucleic acid sequences described above aredisclosed.

A method of identifying a cell having alanine 2,3-aminomutase activityis disclosed. The method includes culturing a cell, which isfunctionally deleted for panD, in media which does not includebeta-alanine nor pantothenate. For example, the cell can producealpha-alanine from media sources of carbon, oxygen, hydrogen, andnitrogen, but which does not include beta-alanine. Cells capable ofgrowing in the media are identified, wherein cell growth indicates thatthe cell is producing beta-alanine from alpha-alanine, which indicatesthe cell has alanine 2,3-aminomutase activity. In contrast, absence ofcell growth indicates that the cell is not producing beta-alanine fromalpha-alanine, which indicates the cell does not have alanine2,3-aminomutase activity. In one example, prior to culturing the cellfor selection, cells are transformed with one or more mutated lysine2,3-aminomutases and/or lysine 5,6-aminomutases.

A method of producing a polypeptide having alanine 2,3-aminomutaseactivity is disclosed. In one example, the method includes culturingcells having at least one exogenous nucleic acid molecule that encodesan alanine 2,3-aminomutase (such as SEQ ID NOS: 20 and 29), which iscapable of producing beta-alanine from alpha-alanine.

Several methods of producing 3-HP from beta-alanine using the disclosedcells having alanine 2,3-aminomutase activity are disclosed. In oneexample, the cell is transfected with one or more enzymes necessary toconvert 3-HP from beta-alanine. In another example, the method includespurifying beta-alanine from the cell, then contacting the beta-alaninewith polypeptides necessary to convert 3-HP from beta-alanine.

The cells, alanine 2,3-aminomutase nucleic and amino acid sequences(such as SEQ ID NOS: 20, 21, 29, and 30), and methods disclosed herein,can be used to produce pantothenate, 3-HP, and derivatives thereof suchas coenzyme A (CoA), and other organic compounds such as1,3-propanediol, acrylic acid, polymerized acrylate, esters of acrylate,polymerized 3-HP, co-polymers of 3-HP and other compounds such asbutyrates, valerates and other compounds, esters of 3-HP, and malonicacid and its esters. 3-HP is both biologically and commerciallyimportant. For example, the nutritional industry can use 3-HP as a food,feed additive or preservative, while the derivatives mentioned above canbe produced from 3-HP.

Nucleic acid molecules encoding for an alanine 2,3-aminomutase (such asSEQ ID NOS: 20 and 29) can be used to engineer host cells with theability to produce 3-HP as well as other organic compounds such as thoselisted above. Alanine 2,3-aminomutase peptides (such as SEQ ID NOS: 21and 30) can be used in cell-free systems to make 3-HP as well as otherorganic compounds such as those listed above. The cells described hereincan be used in culture systems to produce large quantities of 3-HP aswell as other organic compounds such as those listed above.

One aspect of the disclosure provides cells, which in addition toalanine 2,3-aminomutase activity, include other enzyme activities, suchas CoA transferase activity, beta-alanyl-CoA ammonia lyase activity, and3-hydroxypropionyl-CoA dehydratase activity. In addition, methods ofmaking products from these cells are disclosed. In some examples, thecell also includes one or more exogenous nucleic acid molecules thatencodes one or more polypeptides having: glutamate dehydrogenaseactivity, CoA transferase activity, 3-hydroxypropionyl-CoA hydrolase,and/or 3-hydroxyisobutyryl-CoA hydrolase activity, and alaninedehydrogenase or pyruvate-glutamate transaminase activity. In anotherexample, the cell also includes 4-aminobutyrate and/orbeta-alanine-2-oxoglutarate aminotransferase activity and 3-HPdehydrogenase activity and/or 3-hydroxyisobutyrate dehydrogenaseactivity. Additionally, the cell can include, CoA hydrolase activity,poly hydroxyacid synthase activity, and/or lipase or esterase activity.

In another example, a cell including alanine 2,3-aminomutase activity;CoA transferase activity; beta-alanyl-CoA ammonia lyase activity;alanine dehydrogenase or pyruvate-glutamate transaminase activity, and3-HP—CoA dehydratase activity, produces a product, for example, 3-HP,and/or an ester of 3-HP, such as methyl 3-hydroxypropionate, ethyl3-hydroxypropionate, propyl 3-hydroxypropionate, and/or butyl3-hydroxypropionate. In some examples, the cell further includesglutamate dehydrogenase activity, CoA transferase activity,3-hydroxypropionyl-CoA hydrolase, and/or 3-hydroxyisobutyryl-CoAhydrolase activity. Accordingly, the disclosure also provides methods ofproducing one or more of these products. These methods involve culturingthe cell that includes CoA transferase activity; beta-alanyl-CoA ammonialyase activity; 3-HP—CoA dehydratase activity and in some examplesglutamate dehydrogenase activity, CoA transferase activity,3-hydroxypropionyl-CoA hydrolase, 3-hydroxyisobutryl-CoA hydrolaseactivity, and/or alanine dehydrogenase or pyruvate-glutamatetransaminase activity, under conditions that allow the product to beproduced. These cells also can include lipase or esterase activity.

Another aspect of the disclosure provides cells, which in addition toalanine 2,3-aminomutase activity, have CoA transferase activity;beta-alanyl-CoA ammonia lyase activity; 3-hydroxypropionyl-CoAdehydratase activity; and poly hydroxyacid synthase activity. In someexamples, these cells can contain an exogenous nucleic acid moleculethat encodes one or more polypeptides having: CoA transferase activity;beta-alanyl-CoA ammonia lyase activity; 3-hydroxypropionyl-CoAdehydratase activity; alanine dehydrogenase or pyruvate-glutamatetransaminase activity, and poly hydroxyacid synthase activity. This cellcan be used, for example, to produce products such as polymerized 3-HPand co-polymers of 3-HP and other compounds such as butyrates, valeratesand other compounds.

In another example, the cell, which in addition to alanine2,3-aminomutase activity, has CoA transferase activity; beta-alanyl-CoAammonia lyase activity; alanine dehydrogenase or pyruvate-glutamatetransaminase activity, and poly hydroyxacid synthase activity, which canproduce a product, for example, polymerized 3-HP. In some examples,these cells can contain one or more exogenous nucleic acid moleculesthat encode one or more of polypeptides having CoA transferase activity;beta-alanyl-CoA ammonia lyase activity; and/or poly hydroxyacid synthaseactivity.

Another aspect of the disclosure provides a cell including alanine2,3-aminomutase activity, CoA transferase activity, beta-alanyl-CoAammonia lyase activity, alanine dehydrogenase or pyruvate-glutamatetransaminase activity, and lipase or esterase activity. In one example,the cell also includes CoA hydrolase activity. In some examples, thecell contains an exogenous nucleic acid molecule that encodes one ormore polypeptides having CoA transferase activity; beta-alanyl-CoAammonia lyase activity; lipase or esterase activity and/or CoA hydrolaseactivity. This cell can be used, among other things, to produce productssuch as esters of acrylate (e.g., methyl acrylate, ethyl acrylate,propyl acrylate, and butyl acrylate).

Cells which can produce 1,3-propanediol, and methods of their use aredisclosed. 1,3-propanediol can be generated from either 3-HP—CoA or 3-HPvia the use of polypeptides having enzymatic activity. When converting3-HP—CoA to 1,3-propanediol, polypeptides having oxidoreductase activityor reductase activity, such as polypeptides having acetylatingaldehyde:NAD(+) oxidoreductase and alcohol:NAD(+) oxidoreductaseactivities (e.g., enzymes from the 1.1.1.1 and/or 1.2.1.10 class ofenzymes) can be used. When making 1,3-propanediol from 3-HP, acombination of a polypeptide having aldehyde dehydrogenase activity(e.g., an enzyme from the 1.2.1− class) and a polypeptide having alcoholdehydrogenase activity (e.g., an enzyme from the 1.1.1.− class) can beused, such as aldehyde dehydrogenase (NAD(P)+) (EC 1.2.1.−) and alcoholdehydrogenase (EC 1.1.1.1).

In some examples, products are produced in vitro (outside of a cell). Inother examples, products are produced using a combination of in vitroand in vivo (within a cell) methods. In yet other examples, products areproduced in vivo. For methods involving in vivo steps, the cells can beisolated cultured cells or whole organisms such as transgenic plants,non-human mammals, or single-celled organisms such as yeast and bacteria(e.g., Lactobacillus, Lactococcus, Bacillus, and Escherichia cells).Hereinafter such cells are referred to as production cells. Productsproduced by these production cells can be organic products such asbeta-alanine, 3-HP, pantothenate, and derivatives thereof such asorganic acids, polyols (i.e. 1,3-propanediol), coenzyme A (CoA), as wellas an alanine 2,3-aminomutase described herein.

Pantothenate, a vitamin essential to many animals for growth and health,is involved in fatty acid synthesis and degradation. Deficiency of thevitamin results in generalized malaise clinically. Therefore,pantothenate produced using the methods disclosed herein can beadministered to a subject having a pantothenic deficiency, at atherapeutically effective dose. Cells that produce pantothenate, andmethods of producing pantothenate from beta-alanine using the disclosedcells, are disclosed. Production cells used to produce pantothenateand/or CoA, can be used to express alpha-ketopantoatehydroxymethyltransferase (E.C. 2.1.2.11), alpha-ketopantoate reductase(E.C. 1.1.1.169), and pantothenate synthase (E.C. 6.3.2.1), to producepantothenate, or in addition pantothenate kinase (E.C. 2.7.1.33),4′-phosphopantethenoyl-1-cysteine synthetase (E.C. 6.3.2.5),4′-phosphopantothenoylcysteine decarboxylase (E.C. 4.1.1.36),ATP:4′-phosphopantetheine adenyltransferase (E.C. 2.7.7.3), anddephospho-CoA kinase (E.C. 2.7.1.24), to produce coenzyme A.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of a pathway for generating 3-HP and derivativesthereof via a beta-alanine intermediate, and for making beta-alaninefrom alpha-alanine.

FIG. 2 is a diagram of a pathway for generating beta-alanine.

FIG. 3 is a diagram of a pathway for generating coenzyme A andpantothenate from beta-alanine.

FIG. 4 is an alignment of a B. subtilis wild-type lysine 2,3-aminomutase(KAM, SEQ ID NO: 31), and a mutated form thereof which encodes analanine 2,3-aminomutase (SEQ ID NO: 21). Substitutions are shown inbold. The Fe—S cluster-binding motif is underlined, and the putativePLP-binding motif is italicized.

FIG. 5 is an alignment of P. gingivalis wild-type lysine 2,3-aminomutase(kam, SEQ ID NO: 28) and a mutated form thereof which encodes an alanine2,3-aminomutase (aam, SEQ ID NO: 30). Substitutions are shown in bold.The Fe—S cluster-binding motif is underlined, and the putativePLP-binding motif is italicized.

FIG. 6 is an alignment of a B. subtilis and P. gingivalis wild-typelysine 2,3-aminomutase (kam, SEQ ID NOS: 31 and 28), and a mutated formthereof which encodes an alanine 2,3-aminomutase (aam, SEQ ID NOS: 21and 29). Substitutions with a common location are in bold.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NOS: 1 and 2 are PCR primers used to clone a B. subtilis lysine2,3-aminomutase (KAM) gene.

SEQ ID NO: 3 is a nucleic acid sequence of a B. subtilis KAM gene.

SEQ ID NOS: 4 and 5 are PCR primers used to amplify a CAT gene of pKD3.

SEQ ID NOS: 6 and 7 are PCR primers used to confirm correct insertion ofthe CAT gene into the panD locus.

SEQ ID NOS: 8 and 9 are nucleic acid sequences of primers used toamplify the CAT gene of pKD3.

SEQ ID NOS: 10 and 11 are nucleic acid sequences of primers used togenerate an L103M mutation in the wildtype B. subtilis lysine2,3-aminomutase gene.

SEQ ID NOS: 12 and 13 are nucleic acid sequences of primers used togenerate an M136V mutation in the wildtype B. subtilis lysine2,3-aminomutase gene.

SEQ ID NOS: 14 and 15 are nucleic acid sequences of primers used togenerate an D339H mutation in the wildtype B. subtilis lysine2,3-aminomutase gene.

SEQ ID NOS: 16-19, 26, 27 and 32 are nucleic acid sequences of primersused to clone a 3-HP dehydrogenase gene from Alcaligenes faecalis M3A.

SEQ ID NO: 20 is a nucleic acid sequence of an alanine 2,3-aminomutaseDNA.

SEQ ID NO: 21 is an amino acid sequence of an alanine 2,3-aminomutaseprotein.

SEQ ID NO: 22 is a nucleic acid sequence of a beta-alanyl-CoA ammonialyase (ACL-1) cDNA.

SEQ ID NO: 23 is an amino acid sequence of a beta-alanyl-CoA ammonialyase (ACL-1) protein.

SEQ ID NO: 24 is a nucleic acid sequence of a CoA transferase cDNA.

SEQ ID NO: 25 is an amino acid sequence of a CoA transferase protein.

SEQ ID NO: 28 is an amino acid sequence of a P. gingivalis KAM.

SEQ ID NO: 29 is a nucleic acid sequence of an alanine 2,3-aminomutase.

SEQ ID NO: 30 is an amino acid sequence of an alanine 2,3-aminomutaseprotein.

SEQ ID NO: 31 is an amino acid sequence of a B. subtilis KAM.

SEQ ID NO: 33 is an nucleic acid sequence of a 3-HP dehydrogenase genefrom Alcaligenes faecalis M3A.

SEQ ID NO: 34 is an amino acid sequence of a 3-HP dehydrogenase genefrom Alcaligenes faecalis M3A.

SEQ ID NOS: 35-37 are nucleic acid sequences of primers used to clonebeta-alanine-CoA ammonia lyase (ACL-1 and ACL-2).

SEQ ID NOS: 38-40 are nucleic acid sequences of primers used to cloneCoA transferase from E. coli.

SEQ ID NOS: 41-48 are nucleic acid sequences of primers used to generateoperons 1 and 2 which include ACL-1 or ACL-2, CoA transferase and CoAhydratase genes.

SEQ ID NOS: 49-52 are nucleic acid sequences of primers used to generateoperon 3 which includes 4-aminobutyrate aminotransferase and3-hydroxyisobutyrate dehydrogenase genes.

SEQ ID NO: 53 is a nucleic acid sequence of a beta-alanyl-CoA ammonialyase (ACL-2) cDNA.

SEQ ID NO: 54 is an amino acid sequence of a beta-alanyl-CoA ammonialyase (ACL-2) protein.

SEQ ID NOS: 55-56 are nucleic acid sequences of primers used to amplifythe ATH-2 operon from the pATH-2-2-1 plasmid.

SEQ ID NOS: 57-58 are nucleic acid sequences of primers used to amplifythe ATD operon from the pATD plasmid.

SEQ ID NOS: 59-60 are nucleic acid sequences of primers used to amplifya B. subtilis alanine 2,3 aminomutase.

SEQ ID NOS: 61-62 are nucleic acid sequences of primers used to amplifya rat beta-alanine aminotransferase gene.

SEQ ID NOS: 63-64 are nucleic acid sequences of primers used to amplifya 3-HP dehydrogenase from A. faecalis.

SEQ ID NOS: 65-66 are nucleic acid sequences of primers used to amplifyan alpha-alanine aminotransferase gene from rat.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Abbreviations and Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. As used herein,“comprising” means “including” and the singular forms “a” or “an” or“the” include plural references unless the context clearly dictatesotherwise. For example, reference to “comprising a protein” includes oneor a plurality of such proteins, and reference to “comprising the cell”includes reference to one or more cells and equivalents thereof known tothose skilled in the art, and so forth.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features andadvantages of the disclosure are apparent from the following detaileddescription and the claims.

Alanine 2,3-aminomutase: An enzyme which can convert alpha-alanine tobeta-alanine, for example in a cell. Includes any alanine2,3-aminomutase gene, cDNA, RNA, or protein from any organism, such as aprokaryote. In one example, an alanine 2,3-aminomutase is a mutatedlysine 2,3-aminomutase or a mutated lysine 5,6-aminomutase which hasalanine 2,3-aminomutase activity. Lysine 2,3-aminomutases (or genesannotated in genetic databases as lysine 2,3 aminomutase) can beobtained from any organism, such as a prokaryote, for example Bacillussubtilis, Deinococcus radiodurans, Clostridium subterminale,Porphyromonas gingivalis or E. coli, and mutated using any method knownin the art.

In particular examples, an alanine 2,3-aminomutase nucleic acid sequenceincludes the sequence shown in SEQ ID NOS: 20 or 29, or fragments,variants, or fusions thereof that retain the ability to encode a peptideor protein having alanine 2,3-aminomutase activity. In another example,an alanine 2,3-aminomutase protein includes the amino acid sequenceshown in SEQ ID NO: 21 or 30, or fragments, fusions, or variants thereofthat retain alanine 2,3-aminomutase activity.

In another example, an alanine 2,3-aminomutase sequence includes afull-length wild-type sequence, such as SEQ ID NO: 21 or 30, as well asshorter sequences which retain the ability to convert alpha-alanine tobeta-alanine, such as amino acids 50-390 of SEQ ID NO: 21, amino acids101-339 of SEQ ID NO: 21, amino acids 15-390 of SEQ ID NO: 30, and aminoacids 15-340 of SEQ ID NO 30. This description includes alanine2,3-aminomutase allelic variants, as well as any variant, fragment, orfusion sequence which retains the ability to convert alpha-alanine tobeta-alanine.

Alanine 2,3-aminomutase activity: The ability of an alanine2,3-aminomutase to convert alpha-alanine to beta-alanine. In oneexample, such activity occurs in a cell. In another example, suchactivity occurs in vitro. Such activity can be measured using any assayknown in the art, for example the screening assays and enzyme assaysdescribed in EXAMPLES 6 and 9-11. In addition, an enzyme with alanine2,3-aminomutase activity can be identified by incubating the enzyme witheither alpha-alanine or beta-alanine and determining the reactionproducts by high-performance liquid chromatography (for example usingthe method of, Abe et al. J. Chromatography B, 712:43-9, 1998). In oneexample, it is the ability of an alanine 2,3-aminomutase to convertalpha-alanine to beta-alanine in an E. coli mutant functionally deletedfor the panD gene.

Antibody: A molecule including an antigen binding site whichspecifically binds (immunoreacts with) an antigen. Examples includepolyclonal antibodies, monoclonal antibodies, humanized monoclonalantibodies, or immunologically effective portions thereof.

Includes immunoglobulin molecules and immunologically active portionsthereof. Naturally occurring antibodies (e.g., IgG) include fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. However, the antigen-bindingfunction of an antibody can be performed by fragments of a naturallyoccurring antibody. Immunologically effective portions of monoclonalantibodies include, but are not limited to: Fab, Fab′, F(ab′)₂, Fabc andFv portions (for a review, see Better and Horowitz, Methods. Enzymol.1989, 178:476-96). Other examples of antigen-binding fragments include,but are not limited to: (i) an Fab fragment consisting of the VL, VH, CLand CH1 domains; (ii) an Fd fragment consisting of the VH and CH1domains; (iii) an Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (iv) a dAb fragment which consists of a VHdomain; (v) an isolated complimentarily determining region (CDR); and(vi) an F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region. Furthermore,although the two domains of the Fv fragment are coded for by separategenes, a synthetic linker can be made that enables them to be made as asingle protein chain (known as single chain Fv (scFv) by recombinantmethods. Such single chain antibodies are also included.

“Specifically binds” refers to the ability of a particular agent (a“specific binding agent”) to specifically react with a particularanalyte, for example to specifically immunoreact with an antibody, or tospecifically bind to a particular peptide sequence. The binding is anon-random binding reaction, for example between an antibody moleculeand an antigenic determinant. Binding specificity of an antibody istypically determined from the reference point of the ability of theantibody to differentially bind the specific antigen and an unrelatedantigen, and therefore distinguish between two different antigens,particularly where the two antigens have unique epitopes. An antibodythat specifically binds to a particular epitope is referred to as a“specific antibody”.

Monoclonal or polyclonal antibodies can be produced to an alanine2,3-aminomutase polypeptide (such as SEQ ID NO: 21 and/or 30), fragmentsof an alanine 2,3-aminomutase polypeptide (such as amino acids 50-390 ofSEQ ID NO: 21, for example amino acids 101-339 of SEQ ID NO: 21, oramino acids 15-390 of SEQ ID NO: 30, for example amino acids 15-331 ofSEQ ID NO: 30), or variants, fusions, or fragments thereof. Optimally,antibodies raised against one or more epitopes on a polypeptide antigenwill specifically detect that polypeptide. That is, antibodies raisedagainst one particular polypeptide would recognize and bind thatparticular polypeptide, and would not substantially recognize or bind toother polypeptides. The determination that an antibody specificallybinds to a particular polypeptide is made by any one of a number ofstandard immunoassay methods; for instance, Western blotting (See, e.g.,Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989).

To determine that an antibody preparation (such as a preparationproduced in a mouse against an alanine 2,3-aminomutase polypeptide, forexample SEQ ID NO: 21 or 30) specifically detects the appropriatepolypeptide (e.g., an alanine 2,3-aminomutase polypeptide) by Westernblotting, total cellular protein can be extracted from cells andseparated by SDS-polyacrylamide gel electrophoresis. The separated totalcellular protein can then be transferred to a membrane (e.g.,nitrocellulose), and the antibody preparation incubated with themembrane. After washing the membrane to remove non-specifically boundantibodies, the presence of specifically bound antibodies can bedetected using an appropriate secondary antibody (e.g., an anti-mouseantibody) conjugated to an enzyme such as alkaline phosphatase sinceapplication of 5-bromo-4-chloro-3-indolyl phosphate/nitro bluetetrazolium results in the production of a densely blue-colored compoundby immuno-localized alkaline phosphatase.

Substantially pure polypeptides suitable for use as an immunogen can beobtained from transfected cells, transformed cells, or wild-type cells.Polypeptide concentrations in the final preparation can be adjusted, forexample, by concentration on an Amicon filter device, to the level of afew micrograms per milliliter. In addition, polypeptides ranging in sizefrom full-length polypeptides to polypeptides having as few as nineamino acid residues can be utilized as immunogens. Such polypeptides canbe produced in cell culture, can be chemically synthesized usingstandard methods, or can be obtained by cleaving large polypeptides intosmaller polypeptides that can be purified. Polypeptides having as few asnine amino acid residues in length can be immunogenic when presented toan immune system in the context of a Major Histocompatibility Complex(MHC) molecule such as an MHC class I or MHC class II molecule.Accordingly, polypeptides having at least 9, 10, 11, 12, 13, 14, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, or more consecutive amino acid residues ofan alanine 2,3-aminomutase polypeptide can be used as immunogens forproducing antibodies.

Monoclonal antibodies to any of the polypeptides disclosed herein can beprepared from murine hybridomas according to the classic method ofKohler & Milstein (Nature 256:495, 1975) or a derivative method thereof.

Polyclonal antiserum containing antibodies to the heterogeneous epitopesof any polypeptide disclosed herein can be prepared by immunizingsuitable animals with the polypeptide (or fragment, fusion, or variantthereof), which can be unmodified or modified to enhance immunogenicity.An effective immunization protocol for rabbits can be found inVaitukaitis et al. (J. Clin. Endocrinol. Metab. 33:988-91, 1971).

Antibody fragments can be used in place of whole antibodies and can bereadily expressed in prokaryotic host cells. Methods of making and usingimmunologically effective portions of monoclonal antibodies, alsoreferred to as “antibody fragments,” are well known and include thosedescribed in Better & Horowitz (Methods Enzymol. 178:476-96, 1989),Glockshuber et al. (Biochemistry 29:1362-7, 1990), U.S. Pat. No.5,648,237 (“Expression of Functional Antibody Fragments”), U.S. Pat. No.4,946,778 (“Single Polypeptide Chain Binding Molecules”), U.S. Pat. No.5,455,030 (“Immunotherapy Using Single Chain Polypeptide BindingMolecules”), and references cited therein.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are administered, such as injected or absorbed, to ananimal. An antigen reacts with the products of specific humoral orcellular immunity, including those induced by heterologous immunogens.The term “antigen” includes all related antigenic epitopes.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences which determinetranscription. cDNA can be synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Conservative substitution: One or more amino acid substitutions (forexample 1, 2, 5 or 10 residues) for amino acid residues having similarbiochemical properties. Typically, conservative substitutions havelittle to no impact on the activity of a resulting polypeptide. Forexample, a conservative substitution is an amino acid substitution in analanine 2,3-aminomutase peptide that does not substantially affect theability of the peptide to convert alpha-alanine to beta-alanine. In aparticular example, a conservative substitution is an amino acidsubstitution in an alanine 2,3-aminomutase peptide, such as aconservative substitution in SEQ ID NO: 21 or 30, that does notsignificantly alter the ability of the protein to convert alpha-alanineto beta-alanine. Methods that can be used to determine alanine2,3-aminomutase activity are disclosed herein (EXAMPLES 6 and 9-11). Analanine scan can be used to identify which amino acid residues in analanine 2,3-aminomutase peptide can tolerate an amino acid substitution.In one example, alanine 2,3-aminomutase activity is not altered by morethan 25%, for example not more than 20%, for example not more than 10%,when an alanine, or other conservative amino acid (such as those listedbelow), is substituted for one or more native amino acids.

In one example, one conservative substitution is included in thepeptide, such as a conservative substitution in SEQ ID NO: 21 or 30. Inanother example, 10 or less conservative substitutions are included inthe peptide, such as five or less. A polypeptide can be produced tocontain one or more conservative substitutions by manipulating thenucleotide sequence that encodes that polypeptide using, for example,standard procedures such as site-directed mutagenesis or PCR.Alternatively, a polypeptide can be produced to contain one or moreconservative substitutions by using standard peptide synthesis methods.

Substitutional variants are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. Examples of amino acids which may be substituted for anoriginal amino acid in a protein and which are regarded as conservativesubstitutions include: Ser for Ala; Lys for Arg; Gln or His for Asn; Glufor Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro for Gly; Asn or Glnfor His; Leu or Val for Ile; Ile or Val for Leu; Arg or Gln for Lys; Leuor Ile for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyrfor Trp; Trp or Phe for Tyr; and Ile or Leu for Val.

Further information about conservative substitutions can be found in,among other locations in, Ben-Bassat et al., (J. Bacteriol. 169:751-7,1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al.,(Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321-5,1988), WO 00/67796 (Curd et al.) and in standard textbooks of geneticsand molecular biology.

Deletion: The removal of a sequence of a nucleic acid, for example DNA,the regions on either side being joined together.

Detectable: Capable of having an existence or presence ascertained. Forexample, production of beta-alanine from alpha-alanine is detectable ifthe signal generated from the beta-alanine is strong enough to bemeasurable.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid, RNA). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides,referred to as codons, in DNA molecules code for amino acid in apolypeptide. The term codon is also used for the corresponding (andcomplementary) sequences of three nucleotides in the mRNA into which theDNA sequence is transcribed.

Exogenous: The term “exogenous” as used herein with reference to nucleicacid and a particular cell refers to any nucleic acid that does notoriginate from that particular cell as found in nature. Thus, anon-naturally-occurring nucleic acid is considered to be exogenous to acell once introduced into the cell. A nucleic acid that isnaturally-occurring also can be exogenous to a particular cell. Forexample, an entire chromosome isolated from a cell of person X is anexogenous nucleic acid with respect to a cell of person Y once thatchromosome is introduced into Y's cell.

Functional deletion: A mutation, partial or complete deletion,insertion, or other variation made to a gene sequence which inhibitsproduction of the gene product, and/or renders the gene productnon-functional. For example, functional deletion of panD in E. coliprevents the production of β-alanine from aspartate by aspartatedecarboxylase, which is encoded by the panD gene. This functionaldeletion of panD in E. coli inactivates aspartate decarboxylase whichresults in growth inhibition of the E. coli in the absence ofbeta-alanine or pantothenate in the growth medium.

Functionally Equivalent: Having an equivalent function. In the contextof a alanine 2,3-aminomutase molecule, functionally equivalent moleculesinclude different molecules that retain the function of alanine2,3-aminomutase. For example, functional equivalents can be provided bysequence alterations in an alanine 2,3-aminomutase, wherein the peptidewith one or more sequence alterations retains a function of theunaltered peptide, such that it retains its ability to convertalpha-alanine to beta-alanine.

Examples of sequence alterations include, but are not limited to,conservative substitutions, deletions, mutations, frameshifts, andinsertions. In one example, a given polypeptide binds an antibody, and afunctional equivalent is a polypeptide that binds the same antibody.Thus a functional equivalent includes peptides that have the samebinding specificity as a polypeptide, and that can be used as a reagentin place of the polypeptide (such as in the production of pantothenicacid and 3-HP). In one example a functional equivalent includes apolypeptide wherein the binding sequence is discontinuous, wherein theantibody binds a linear epitope. Thus, if the peptide sequence isMKNKWYKPKR (amino acids 1-10 of SEQ ID NO: 21) a functional equivalentincludes discontinuous epitopes, that can appear as follows (**=anynumber of intervening amino acids):NH₂—**-M**K**N**K**W**Y**K**P**K**R—COOH. In this example, thepolypeptide is functionally equivalent to amino acids 1-10 of SEQ ID NO:21 if the three dimensional structure of the polypeptide is such that itcan bind a monoclonal antibody that binds amino acids 1-10 of SEQ ID NO:21.

Hybridization: A method of testing for complementarity in the nucleotidesequence of two nucleic acid molecules, based on the ability ofcomplementary single-stranded DNA and/or RNA to form a duplex molecule.Nucleic acid hybridization techniques can be used to obtain an isolatednucleic acid within the scope of the disclosure. Briefly, any nucleicacid having some homology to an alanine 2,3-aminomutase (such ashomology to SEQ ID NOS: 20 and 29 or variants or fragments thereof) canbe used as a probe to identify a similar nucleic acid by hybridizationunder conditions of moderate to high stringency. Once identified, thenucleic acid then can be purified, sequenced, and analyzed to determineif it is an alanine 2,3-aminomutase having alanine 2,3-aminomutaseactivity.

Hybridization can be done by Southern or Northern analysis to identify aDNA or RNA sequence, respectively, that hybridizes to a probe. The probecan be labeled, for example with a biotin, a fluorophore, digoxygenin,an enzyme, or a radioisotope such as ³²P. The DNA or RNA to be analyzedcan be electrophoretically separated on an agarose or polyacrylamidegel, transferred to nitrocellulose, nylon, or other suitable membrane,and hybridized with the probe using standard techniques well known inthe art such as those described in sections 7.39-7.52 of Sambrook etal., (1989) Molecular Cloning, second edition, Cold Spring HarborLaboratory, Plainview, N.Y. Typically, a probe is at least about 20nucleotides in length. For example, a probe including 20 contiguousnucleotides of an alanine 2,3-aminomutase (such as 20 contiguousnucleotides of SEQ ID NO: 20 or 29) can be used to identify an identicalor similar nucleic acid. In addition, probes longer or shorter than 20nucleotides can be used.

The disclosure also provides isolated nucleic acid sequences that are atleast about 12 bases in length (e.g., at least about 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 40, 50, 60, 100, 250, 500, 750, 1000, 1400, 2000,3000, 4000, or 5000 bases in length) and hybridize, under hybridizationconditions, to the sense or antisense strand of an alanine2,3-aminomutase nucleic acid sequence, for example SEQ ID NO: 20 or 29).The hybridization conditions can be moderately or highly stringenthybridization conditions.

Moderately stringent hybridization conditions are when the hybridizationis performed at about 42° C. in a hybridization solution containing 25mM KPO₄ (pH 7.4), 5×SSC, 5×Denhart's solution, 50 μg/mL denatured,sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15ng/mL probe (about 5×10⁷ cpm/μg), while the washes are performed atabout 50° C. with a wash solution containing 2×SSC and 0.1% sodiumdodecyl sulfate.

Highly stringent hybridization conditions are when the hybridization isperformed at about 42° C. in a hybridization solution containing 25 mMKPO₄ (pH 7.4), 5×SSC, 5×Denhart's solution, 50 μg/mL denatured,sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15ng/mL probe (about 5×10⁷ cpm/μg), while the washes are performed atabout 65° C. with a wash solution containing 0.2×SSC and 0.1% sodiumdodecyl sulfate.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule or protein) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, i.e., other chromosomal andextrachromosomal DNA and RNA, and proteins. Nucleic acids and proteinsthat have been “isolated” include nucleic acids and proteins purified bystandard purification methods. The term also embraces nucleic acids andproteins prepared by recombinant expression in a host cell as well aschemically synthesized nucleic acids, proteins and peptides.

In one example, isolated refers to a naturally-occurring nucleic acidthat is not immediately contiguous with both of the sequences with whichit is immediately contiguous (one on the 5′ end and one on the 3′ end)in the naturally-occurring genome of the organism from which it isderived. For example, an isolated nucleic acid can be, withoutlimitation, a recombinant DNA molecule of any length, provided one ofthe nucleic acid sequences normally found immediately flanking thatrecombinant DNA molecule in a naturally-occurring genome is removed orabsent. Thus, an isolated nucleic acid includes, without limitation, arecombinant DNA that exists as a separate molecule (e.g., a cDNA or agenomic DNA fragment produced by PCR or restriction endonucleasetreatment) independent of other sequences as well as recombinant DNAthat is incorporated into a vector, an autonomously replicating plasmid,a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into thegenomic DNA of a prokaryote or eukaryote. In addition, an isolatednucleic acid can include a recombinant DNA molecule that is part of ahybrid or fusion nucleic acid sequence.

In one example, the term “isolated” as used with reference to nucleicacid also includes any non-naturally-occurring nucleic acid sincenon-naturally-occurring nucleic acid sequences are not found in natureand do not have immediately contiguous sequences in anaturally-occurring genome. For example, non-naturally-occurring nucleicacid such as an engineered nucleic acid is considered to be isolatednucleic acid. Engineered nucleic acid can be made using common molecularcloning or chemical nucleic acid synthesis techniques. Isolatednon-naturally-occurring nucleic acid can be independent of othersequences, or incorporated into a vector, an autonomously replicatingplasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), orthe genomic DNA of a prokaryote or eukaryote. In addition, anon-naturally-occurring nucleic acid can include a nucleic acid moleculethat is part of a hybrid or fusion nucleic acid sequence.

Leucine 2,3-aminomutase: An enzyme which can convert alpha-leucine tobeta-leucine. Includes any leucine 2,3-aminomutase gene, cDNA, RNA, orprotein from any organism, such as a prokaryote or eukaryote, forexample from rat, human, chicken, or Clostridium sporogenes (Poston, J.Biol. Chem. 251:1859-63, 1976). This description includes leucine2,3-aminomutase allelic variants, as well as any variant, fragment, orfusion protein sequence which retains the ability to convertalpha-leucine to beta-leucine.

Lysine 2,3-aminomutase: An enzyme which can convert alpha-lysine tobeta-lysine. Includes any lysine 2,3-aminomutase gene, cDNA, RNA, orprotein from any organism, such as a prokaryote, for example Bacillussubtilis, Deinococcus radiodurans, Clostridium subterminale,Porphyromonas gingivalis, Aquifex aeolicus, Haemophilus influenzae, orE. coli. This description includes lysine 2,3-aminomutase allelicvariants, as well as any variant, fragment, or fusion sequence whichretains the ability to convert alpha-lysine to beta-lysine. In oneexample, includes polypeptides encoded by genes annotated as lysine2,3-aminomutase in public DNA sequence databases, such as GenBank.

Nucleic acid: Encompasses both RNA and DNA including, withoutlimitation, cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. In addition, nucleic acid can becircular or linear.

Oligonucleotide: A linear polynucleotide (such as DNA or RNA) sequenceof at least 9 nucleotides, for example at least 15, 18, 24, 25, 27, 30,50, 100 or even 200 nucleotides long.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Pantothenate or Pantothenic Acid: A commercially significant vitaminwhich is used in cosmetics, medicine, and nourishment. The termspantothenic acid and pantothenate are used interchangeably herein, andrefer not only to the free acid but also to the salts of D-pantothenicacid, such as the calcium salt, sodium salt, ammonium salt or potassiumsalt. Pantothenate can be produced by chemical synthesis orbiotechnologically from beta-alanine using the cells and methodsdisclosed herein.

Methods for measuring the amount of pantothenate are known (for examplesee U.S. Pat. No. 6,184,006 to Rieping et al. and U.S. Pat. No.6,177,264 to Eggeling et al.). For example, a quantitative determinationof D-pantothenate can be made by using the Lactobacillus plantarumpantothenate assay (test strain: Lactobacillus plantarum ATCC 8014, Cat.No. 3211-30-3; culture medium: Bacto pantothenate assay medium (DIFCOLaboratories, Michigan, USA), cat. No. 0604-15-3). This indicator straincan grow only in the presence of pantothenate in the indicated culturemedium and displays a photometrically measurable, linear dependency ofthe growth on the concentration of pantothenate in the medium. Thehemicalcium salt of pantothenate can be used for calibration (SigmaCatalog Number P 2250). The optical density can be determined at awavelength of 580 nm.

Peptide Modifications: The present disclosure includes alanine2,3-aminomutase peptides, as well as synthetic embodiments. In addition,analogues (non-peptide organic molecules), derivatives (chemicallyfunctionalized peptide molecules obtained starting with the disclosedpeptide sequences) and variants (homologs) having alanine2,3-aminomutase activity can be utilized in the methods describedherein. The peptides disclosed herein include a sequence of amino acids,that can be either L- and/or D-amino acids, naturally occurring andotherwise.

Peptides can be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedpeptides, and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, may be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the peptide,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains may be substituted with one ormore halogen atoms, such as F, Cl, Br or I, or with C₁-C₁₆ alkyl, C₁-C₁₆alkoxy, carboxylic acids and esters thereof, or amides of suchcarboxylic acids. Methylene groups of the peptide side chains can beextended to homologous C₂-C₄ alkylenes. Thiols can be protected with anyone of a number of well-recognized protecting groups, such as acetamidegroups. Those skilled in the art will also recognize methods forintroducing cyclic structures into the peptides of this disclosure toselect and provide conformational constraints to the structure thatresult in enhanced stability. For example, a C- or N-terminal cysteinecan be added to the peptide, so that when oxidized the peptide willcontain a disulfide bond, generating a cyclic peptide. Other peptidecyclizing methods include the formation of thioethers and carboxyl- andamino-terminal amides and esters.

Peptidomimetic and organomimetic embodiments are also within the scopeof the present disclosure, whereby the three-dimensional arrangement ofthe chemical constituents of such peptido- and organomimetics mimic thethree-dimensional arrangement of the peptide backbone and componentamino acid side chains, resulting in such peptido- and organomimetics ofthe proteins of this invention having detectable alanine 2,3-aminomutaseactivity. For computer modeling applications, a pharmacophore is anidealized, three-dimensional definition of the structural requirementsfor biological activity. Peptido- and organomimetics can be designed tofit each pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs”, in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptionsof techniques used in CADD. Also included within the scope of thedisclosure are mimetics prepared using such techniques. In one example,a mimetic mimics the alanine 2,3-aminomutase activity generated by analanine 2,3-aminomutase or a variant, fragment, or fusion thereof.

Polynucleotide: A linear nucleic acid sequence of any length. Therefore,a polynucleotide includes molecules which are at least about 15, 25, 50,75, 100, 200 or 400 (oligonucleotides) and also nucleotides as long as afull-length cDNA.

Probes and primers: A “probe” includes an isolated nucleic acidcontaining a detectable label or reporter molecule. Typical labelsinclude radioactive isotopes, ligands, chemiluminescent agents,fluorophores, and enzymes. Methods for labeling and guidance in thechoice of labels appropriate for various purposes are discussed in, forexample, Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989, and Ausubel et al. (ed.) Current Protocols inMolecular Biology, Greene Publishing and Wiley-Interscience, New York(with periodic updates), 1987.

“Primers” are typically nucleic acid molecules having ten or morenucleotides (e.g., nucleic acid molecules having between about 10nucleotides and about 100 nucleotides). A primer can be annealed to acomplementary target nucleic acid strand by nucleic acid hybridizationto form a hybrid between the primer and the target nucleic acid strand,and then extended along the target nucleic acid strand by, for example,a DNA polymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, for example, by the polymerase chain reaction(PCR) or other nucleic-acid amplification methods.

Methods for preparing and using probes and primers are described, forexample, in references such as Sambrook et al. (ed.), Molecular Cloning:A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989; Ausubel et al. (ed.), CurrentProtocols in Molecular Biology, Greene Publishing andWiley-Interscience, New York (with periodic updates), 1987; and Innis etal., PCR Protocols: A Guide to Methods and Applications, Academic Press:San Diego, 1990. PCR primer pairs can be derived from a known sequence,for example, by using computer programs intended for that purpose suchas Primer (Version 0.5, © 1991, Whitehead Institute for BiomedicalResearch, Cambridge, Mass.). One of skill in the art will appreciatethat the specificity of a particular probe or primer increases with thelength, but that a probe or primer can range in size from a full-lengthsequence to sequences as short as five consecutive nucleotides. Thus,for example, a primer of 20 consecutive nucleotides can anneal to atarget with a higher specificity than a corresponding primer of only 15nucleotides. Thus, in order to obtain greater specificity, probes andprimers can be selected that comprise, for example, 25, 30, 35, 40, 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1450, 1500, or more consecutive nucleotides.

Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of a polymerase II type promoter, a TATA element. A promoter alsooptionally includes distal enhancer or repressor elements which can belocated as much as several thousand base pairs from the start site oftranscription.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its environment within a cell, such thatthe peptide is substantially separated from cellular components (nucleicacids, lipids, carbohydrates, and other polypeptides) that may accompanyit. In another example, a purified peptide preparation is one in whichthe peptide is substantially-free from contaminants, such as those thatmight be present following chemical synthesis of the peptide.

In one example, an alanine 2,3-aminomutase peptide is purified when atleast 50% by weight of a sample is composed of the peptide, for examplewhen at least 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% or more ofa sample is composed of the peptide. Examples of methods that can beused to purify an antigen, include, but are not limited to the methodsdisclosed in Sambrook et al. (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, N.Y., 1989, Ch. 17). Protein purity can bedetermined by, for example, polyacrylamide gel electrophoresis of aprotein sample, followed by visualization of a single polypeptide bandupon staining the polyacrylamide gel; high-pressure liquidchromatography; sequencing; or other conventional methods.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring and/or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques. Recombinantis also used to describe nucleic acid molecules that have beenartificially manipulated, but contain the same regulatory sequences andcoding regions that are found in the organism from which the nucleicacid was isolated.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Sequencesimilarity can be measured in terms of percentage similarity (whichtakes into account conservative amino acid substitutions); the higherthe percentage, the more similar the sequences are. Homologs ororthologs of nucleic acid or amino acid sequences possess a relativelyhigh degree of sequence identity/similarity when aligned using standardmethods. This homology is more significant when the orthologous proteinsor cDNAs are derived from species which are more closely related (e.g.,human and mouse sequences), compared to species more distantly related(e.g., human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

BLASTN is used to compare nucleic acid sequences, while BLASTP is usedto compare amino acid sequences. To compare two nucleic acid sequences,the options can be set as follows: −i is set to a file containing thefirst nucleic acid sequence to be compared (e.g., C:\seq1.txt); −j isset to a file containing the second nucleic acid sequence to be compared(e.g., C:\seq2.txt); −p is set to blastn; −o is set to any desired filename (e.g., C:\output.txt); −q is set to −1; −r is set to 2; and allother options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two sequences: C:\B12seq −i c:\seq1.txt −jc:\seq2.txt −p blastn −o c:\output.txt −q −1 −r 2.

To compare two amino acid sequences, the options of B12seq can be set asfollows: −i is set to a file containing the first amino acid sequence tobe compared (e.g., C:\seq1.txt); −j is set to a file containing thesecond amino acid sequence to be compared (e.g., C:\seq2.txt); −p is setto blastp; −o is set to any desired file name (e.g., C:\output.txt); andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two amino acid sequences: C:\B12seq −i c:\seq1.txt −jc:\seq2.txt −p blastp −o c:\output.txt. If the two compared sequencesshare homology, then the designated output file will present thoseregions of homology as aligned sequences. If the two compared sequencesdo not share homology, then the designated output file will not presentaligned sequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (e.g.,100 consecutive nucleotides or amino acid residues from a sequence setforth in an identified sequence), followed by multiplying the resultingvalue by 100. For example, a nucleic acid sequence that has 1166 matcheswhen aligned with a test sequence having 1154 nucleotides is 75.0percent identical to the test sequence (i.e., 1166÷1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer. In another example, a target sequencecontaining a 20-nucleotide region that aligns with 20 consecutivenucleotides from an identified sequence as follows contains a regionthat shares 75 percent sequence identity to that identified sequence(i.e., 15÷20*100=75).

For comparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). Homologs are typically characterizedby possession of at least 70% sequence identity counted over thefull-length alignment with an amino acid sequence using the NCBI BasicBlast 2.0, gapped blastp with databases such as the nr or swissprotdatabase. Queries searched with the blastn program are filtered withDUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70).Other programs use SEG. In addition, a manual alignment can beperformed. Proteins with even greater similarity will show increasingpercentage identities when assessed by this method, such as at least75%, 80%, 85%, 90%, 95%, or 99% sequence identity.

When aligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequence will show increasing percentage identities whenassessed by this method, such as at least 60%, 70%, 75%, 80%, 85%, 90%,95%, 98%, 99% sequence identity. When less than the entire sequence isbeing compared for sequence identity, homologs will typically possess atleast 75% sequence identity over short windows of 10-20 amino acids, andcan possess sequence identities of at least 85%, 90%, 95% or 98%depending on their identity to the reference sequence. Methods fordetermining sequence identity over such short windows are described atthe NCBI web site.

One indication that two nucleic acid molecules are closely related isthat the two molecules hybridize to each other under stringentconditions. Stringent conditions are sequence-dependent and aredifferent under different environmental parameters. Nucleic acidmolecules that hybridize under stringent conditions to an alanine2,3-aminomutase gene sequence typically hybridize to a probe based oneither an entire alanine 2,3-aminomutase gene or selected portions ofthe gene, respectively, under conditions described above.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode identical or similar (conserved) amino acidsequences, due to the degeneracy of the genetic code. Changes in anucleic acid sequence can be made using this degeneracy to producemultiple nucleic acid molecules that all encode substantially the sameprotein. Such homologous nucleic acid sequences can, for example,possess at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identitydetermined by this method.

One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is possible that stronglysignificant homologs could be obtained that fall outside the rangesprovided.

An alternative (and not necessarily cumulative) indication that twonucleic acid sequences are substantially identical is that thepolypeptide which the first nucleic acid encodes is immunologicallycross reactive with the polypeptide encoded by the second nucleic acid.

Specific binding agent: An agent that binds substantially only to adefined target, such as a peptide target. For example, an alanine2,3-aminomutase binding agent includes anti-alanine 2,3-aminomutaseantibodies and other agents (such as peptide or drugs) that bindsubstantially to only an alanine 2,3-aminomutase. Antibodies to analanine 2,3-aminomutase protein (or fragments thereof) can be used topurify or identify such a protein.

Transformed: A cell into which a nucleic acid molecule has beenintroduced, for example by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, including, but notlimited to transfection with viral vectors, conjugation, transformationwith plasmid vectors, and introduction of naked DNA by electroporation,lipofection, and particle gun acceleration.

Variants, fragments or fusion proteins: The disclosed alanine 2,3aminomutase proteins, include variants, fragments, and fusions thereof.DNA sequences which encode for a protein (for example SEQ ID NO: 20 or29), fusion alanine 2,3 aminomutase protein, or a fragment or variant ofan alanine 2,3 aminomutase protein, can be engineered to allow theprotein to be expressed in eukaryotic cells, bacteria, insects, and/orplants. To obtain expression, the DNA sequence can be altered andoperably linked to other regulatory sequences. The final product, whichcontains the regulatory sequences and the protein, is referred to as avector. This vector can be introduced into eukaryotic, bacteria, insect,and/or plant cells. Once inside the cell the vector allows the proteinto be produced.

A fusion protein including a protein, such as an alanine 2,3-aminomutase(or variant, polymorphism, mutant, or fragment thereof), for example SEQID NO: 21 or 30, linked to other amino acid sequences that do notinhibit the desired activity of alanine 2,3-aminomutase, for example theability to convert alpha-alanine to beta-alanine. In one example, theother amino acid sequences are no more than about 10, 12, 15, 20, 25,30, or 50 amino acids in length.

One of ordinary skill in the art will appreciate that a DNA sequence canbe altered in numerous ways without affecting the biological activity ofthe encoded protein. For example, PCR can be used to produce variationsin the DNA sequence which encodes an alanine 2,3-aminomutase. Suchvariants can be variants optimized for codon preference in a host cellused to express the protein, or other sequence changes that facilitateexpression.

Vector: A nucleic acid molecule as introduced into a cell, therebyproducing a transformed cell. A vector may include nucleic acidsequences that permit it to replicate in the cell, such as an origin ofreplication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Alanine 2,3-Aminomutase Nucleic Acids and Polypeptides

Polypeptides having alanine 2,3-aminomutase activity are disclosedherein. In one example, the polypeptide is a mutated aminomutase aminoacid sequence, such as a lysine 2,3-aminomutase, leucine2,3-aminomutase, or lysine 5,6-aminomutase sequence. Examples of apolypeptide having alanine 2,3-aminomutase activity are shown in SEQ IDNOS: 21 and 30. However, the disclosure also encompasses variants,fusions, and fragments of SEQ ID NOS: 21 and 30 which retain alanine2,3-aminomutase activity. Examples of fragments which can be usedinclude, but are not limited to: amino acids 50-390, 50-350, 60-350,75-340, or 100-339 of SEQ ID NO: 21 and amino acids 1-390, 15-390,15-340 or 19-331 of SEQ ID NO:30. Examples of substitutions which can bemade, while still retaining alanine 2,3-aminomutase activity, include,but are not limited to: V21I or V21L; Y71P; L171; K361R; A410V; and/orY430F or Y430W of SEQ ID NO: 21, and T40S; V96I or V96L; D102E; A252V;and/or L393V of SEQ ID NO: 30, as well as combinations thereof.

The disclosure provides enzyme polypeptides, such as an alanine2,3-aminomutase (for example SEQ ID NO: 21 and/or 30, and variants,fragments, and fusions thereof that retain alanine 2,3-aminomutaseactivity). One skilled in the art will understand that variant enzymesequences can be used, as long as the enzyme retains the desired enzymeactivity, such as alanine 2,3-aminomutase activity. For example, thedisclosure provides polypeptides that contain at least 15 contiguousamino acids which are identical to an enzyme sequence, such as analanine 2,3-aminomutase sequence. It will be appreciated that thedisclosure also provides polypeptides that contain an amino acidsequence that is greater than at least 15 amino acid residues (e.g., atleast 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50,75, 100, 150, 200, 250, 300 or more amino acid residues) and identicalto any enzyme disclosed herein or otherwise publicly available.

In addition, the disclosure provides enzyme polypeptides, such as analanine 2,3-aminomutase peptide (e.g. SEQ ID NO: 21 and/or 30), whichincludes an amino acid sequence having a variation of the enzyme aminoacid sequence. Variant sequences can contain a single insertion, asingle deletion, a single substitution, multiple insertions, multipledeletions, multiple substitutions, or any combination thereof (e.g.,single deletion together with multiple insertions). Such polypeptidesshare at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99% sequenceidentity with an enzyme sequence, such as an alanine 2,3-aminomutasesequence, as long as the peptide encoded by the amino acid sequenceretains the desired enzyme activity.

Polypeptides having a variant amino acid sequence can retain enzymaticactivity, such as alanine 2,3-aminomutase activity. Such polypeptidescan be produced by manipulating the nucleotide sequence encoding apolypeptide using standard procedures such as site-directed mutagenesisor PCR. One type of modification includes the substitution of one ormore amino acid residues, such as no more than 10 amino acids, for aminoacid residues having a similar biochemical property, that is, aconservative substitution.

More substantial changes can be obtained by selecting substitutions thatare less conservative, e.g., selecting residues that differ moresignificantly in their effect on maintaining: (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation; (b) the charge or hydrophobicity of thepolypeptide at the target site; or (c) the bulk of the side chain. Thesubstitutions that in general are expected to produce the greatestchanges in polypeptide function are those in which: (a) a hydrophilicresidue, e.g., serine or threonine, is substituted for (or by) ahydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine oralanine; (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g.,lysine, arginine, or histidine, is substituted for (or by) anelectronegative residue, e.g., glutamic acid or aspartic acid; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine. The effects ofthese amino acid substitutions (or other deletions or additions) can beassessed for polypeptides having enzymatic activity by analyzing theability of the polypeptide to catalyze the conversion of the samesubstrate as the related native polypeptide to the same product as therelated native polypeptide. Accordingly, polypeptides having no morethan 5, 10, 20, 30, 40, or 50 conservative substitutions are providedherein.

Also disclosed are isolated nucleic acids that encode polypeptideshaving alanine 2,3-aminomutase activity, for example a sequence whichincludes SEQ ID NO: 20 or 29. However, the disclosure also encompassesvariants, fusions, and fragments of SEQ ID NOS: 20 and 29 which retainthe ability to encode a protein or peptide having alanine2,3-aminomutase activity. In one example an isolated nucleic acidencoding a polypeptide having alanine 2,3-aminomutase activity isoperably linked to a promoter sequence, and can be part of a vector. Thenucleic acid can be a recombinant nucleic acid, that can be used totransform cells and make transformed cells and/or transgenic non-humanmammals.

Transformed cells including at least one exogenous nucleic acid moleculewhich encodes a polypeptide having alanine 2,3-aminomutase activity(such as SEQ ID NO: 20 and/or 29 or fragments, fusions, or variantsthereof that retain alanine 2,3-aminomutase activity), is disclosed. Inone example, such a transformed cell produces beta-alanine fromalpha-alanine. In another example, the cell produces 3-HP, pantothenate,CoA, and/or organic compounds such as 1,3-propanediol.

The nucleic acid sequences encoding the enzymes disclosed herein, suchas alanine 2,3-aminomutase (SEQ ID NO: 20 and 29), lysine2,3-aminomutase (SEQ ID NOS: 3 and 28), and beta-alanyl-CoA ammonialyase (SEQ ID NO: 22), (as well as any other enzyme disclosed herein),can contain an entire nucleic acid sequence encoding the enzyme, as wellas a portions thereof that retain the desired enzyme activity. Forexample, an enzyme nucleic acid can contain at least 15 contiguousnucleotides of an enzyme nucleic acid sequence. It will be appreciatedthat the disclosure also provides isolated nucleic acid that contains anucleotide sequence that is greater than 15 nucleotides (e.g., at least16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 75,10, 200, 500 or more nucleotides) in length and identical to any portionof an enzyme sequence, such as an alanine 2,3-aminomutase sequence shownin SEQ ID NO: 20 and/or 29.

In addition, the disclosure provides isolated enzyme nucleic acidsequences which contains a variation of an enzyme sequence, such as avariant alanine 2,3-aminomutase nucleic acid sequence. Variants cancontain a single insertion, a single deletion, a single substitution,multiple insertions, multiple deletions, multiple substitutions, or anycombination thereof (e.g., single deletion together with multipleinsertions) as long as the peptide encoded thereby retains alanine2,3-aminomutase activity. Such isolated nucleic acid molecules can shareat least 60, 70, 75, 80, 85, 90, 92, 95, 97, 98, or 99% sequenceidentity with an enzyme sequence, such as an alanine 2,3-aminomutasesequence, as long as the peptide encoded by the nucleic acid retains thedesired enzyme activity, such as alanine 2,3-aminomutase activity. Forexample, the following variations can be made to the alanine2,3-aminomutase nucleic acid sequence: for SEQ ID NO: 20, the “a” atposition 12 can be substituted with an “g”; the “g” at position 1050 canbe substituted with an “a”; the “a” at position 255; can be substitutedwith an “g” “t” or “c;” for SEQ ID NO: 29, the “a” at position 6 can besubstituted with a “g” “t” or “c”; the “t” at position 66 can besubstituted with a “c”; and the “g” at position 315; can be substitutedwith an “a” “t” or c.

Codon preferences and codon usage tables for a particular species can beused to engineer isolated nucleic acid molecules that take advantage ofthe codon usage preferences of that particular species. For example, theenzymes disclosed herein can be designed to have codons that arepreferentially used by a particular organism of interest.

The disclosure also provides isolated nucleic acid sequences that encodefor an enzyme, such as alanine 2,3-aminomutase, wherein the sequence isat least about 12 bases in length (e.g., at least about 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 40, 50, 60, 100, 250, 500, 750, 1000, 1500,2000, 3000, 4000, or 5000 bases in length) and hybridizes, underhybridization conditions, to the sense or antisense strand of a nucleicacid encoding the enzyme. The hybridization conditions can be moderatelyor highly stringent hybridization conditions.

Polypeptides and nucleic acid encoding polypeptide can be produced bystandard DNA mutagenesis techniques, for example, M13 primermutagenesis. Details of these techniques are provided in Sambrook et al.(ed.), Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, ColdSpring Harbor Laboratory Press, Cold Spring, Harbor, N.Y., 1989, Ch. 15.Nucleic acid molecules can contain changes of a coding region to fit thecodon usage bias of the particular organism into which the molecule isto be introduced.

Alternatively, the coding region can be altered by taking advantage ofthe degeneracy of the genetic code to alter the coding sequence in sucha way that, while the nucleic acid sequence is substantially altered, itnevertheless encodes a polypeptide having an amino acid sequenceidentical or substantially similar to the native amino acid sequence.For example, because of the degeneracy of the genetic code, alanine isencoded by the four nucleotide codon triplets: GCT, GCA, GCC, and GCG.Thus, the nucleic acid sequence of the open reading frame can be changedat an alanine position to any of these codons without affecting theamino acid sequence of the encoded polypeptide or the characteristics ofthe polypeptide. Based upon the degeneracy of the genetic code, nucleicacid variants can be derived from a nucleic acid sequence using astandard DNA mutagenesis techniques as described herein, or by synthesisof nucleic acid sequences. Thus, this disclosure also encompassesnucleic acid molecules that encode the same polypeptide but vary innucleic acid sequence by virtue of the degeneracy of the genetic code.

Cells with Alanine 2,3-Aminomutase Activity

Cells having alanine 2,3-aminomutase activity are disclosed. Such cellscan produce beta-alanine from alpha-alanine. In one example, such cellshave alanine 2,3-aminomutase activity due to a naturally occurringmutation, and/or a mutation induced in the chromosome(s) of the cell,for example by exposing the cell to chemical or UV mutagenesis. Cellsincluding alanine 2,3-aminomutase activity can be eukaryotic orprokaryotic. Examples of such cells include, but are not limited toLactobacillus, Lactococcus, Bacillus, Escherichia, Geobacillus,Corynebacterium, Clostridium, fungal, plant, and yeast cells. In oneexample, a plant cell is part of a plant, such as a transgenic plant.

In one example, cells having alanine 2,3-aminomutase activity aretransformed cells. Such cells can include at least one exogenous nucleicacid molecule that encodes an alanine 2,3-aminomutase, for example asequence comprising SEQ ID NO: 20 or 29, or variants, fragments, orfusions thereof that retain the ability to encode a protein havingalanine 2,3-aminomutase activity. In one example, the exogenous nucleicacid molecule is a mutated lysine 2,3-aminomutase, such as a mutatedprokaryotic lysine 2,3-aminomutase. In specific examples, the mutatedprokaryotic lysine 2,3-aminomutase is a mutated Bacillus subtilis,Deinococcus radiodurans, Clostridium subterminale, Aquifex aeolicus,Haemophilus influenzae, E. coli, or Porphyromonas gingivalis lysine2,3-aminomutase. Other lysine 2,3-aminomutases can be identified byusing methods known in the art, for example by searching for similarsequences on BLAST and/or by using hybridization methods. In a specificexample, the mutated lysine 2,3-aminomutase is a mutated B. subtilis ora mutated P. gingivalis lysine 2,3-aminomutase. In an another example,the exogenous nucleic acid molecule is a mutated lysine 5,6-aminomutase,such as a mutated prokaryotic lysine 5,6-aminomutase. Alternatively, theexogenous nucleic acid molecule is a mutated leucine 2,3-aminomutase, ora mutated lysine 5,6-aminomutase, such as a mutated C. sticklandiilysine 5,6-aminomutase.

In a particular example, the mutated lysine 2,3-aminomutase is a mutatedB. subtilis lysine 2,3-aminomutase having a substitution at positionL103, D339 and/or M136. For example, the substitution can include aL103M, L103K, L103R, L103E, or L103S substitution. In another oradditional example, the substitution includes a D339H, D339Q, D339T, orD339N substitution. In yet another example, the substitution can includea L103M, a M136V substitution, a D339H substitution, or any combinationthereof.

Cells which include alanine 2,3-aminomutase activity as well as otherenzyme activities, are disclosed. Such cells can be used to producebeta-alanine, 3-HP, pantothenate, CoA, and organic acids, polyols suchas 1,3-propanediol, acrylic acid, polymerized acrylate, esters ofacrylate, polymerized 3-HP, co-polymers of 3-HP and other compounds suchas butyrates, valerates and other compounds, and esters of 3-HP.

In one example, such cells also include alanine dehydrogenase orpyruvate/glutamate transaminase activity, CoA transferase activity orCoA synthetase, beta-alanyl-CoA ammonia lyase activity, 3-HP—CoAdehydratase activity, glutamate dehydrogenase activity,3-hydroxypropionyl-CoA hydrolase, or 3-hydroxyisobutryl-CoA hydrolaseactivity. In another example, such cells also include alaninedehydrogenase or pyruvate-glutamate transaminase activity,4-aminobutyrate and/or beta-alanine-2-oxoglutarate aminotransferaseactivity, glutamate dehydrogenase activity, and 3-HP or3-hydroxyisobutyrate dehydrogenase activity. In these examples, thecells can be used to produce 3-HP.

In another example, the cells also include alanine dehydrogenase orpyruvate/glutamate transaminase activity, CoA transferase or CoAsynthetase activity, beta-alanyl-CoA ammonia lyase activity, 3-HP—CoAdehydratase activity, glutamate dehydrogenase activity, and3-hydroxypropionyl-CoA hydrolase or 3-hydroxyisobutryl-CoA hydrolaseactivity. In another example, such cells also include alaninedehydrogenase or pyruvate-glutamate transaminase activity,4-aminobutyrate and/or beta-alanine-2-oxoglutarate aminotransferaseactivity, glutamate dehydrogenase activity, and 3-HP or3-hydroxyisobutyrate dehydrogenase activity; and lipase or esteraseactivity. Such cells can be used to produce an ester of 3-HP, such asmethyl 3-hydroxypropionate, ethyl 3-hydroxypropionate, propyl3-hydroxypropionate, butyl 3-hydroxypropionate, or 2-ethylhexyl3-hydroxypropionate.

In another example, the cells also include alanine dehydrogenase orpyruvate/glutamate transaminase activity, CoA synthetase activity,beta-alanyl-CoA ammonia lyase activity, 3-HP—CoA dehydratase activity,glutamate dehydrogenase activity; and poly hydroxacid synthase activity.Such cells can be used to produce polymerized 3-HP.

In yet another example, the cells also include alanine dehydrogenase orpyruvate/glutamate transaminase activity, CoA synthetase activity,beta-alanyl-CoA ammonia lyase activity, glutamate dehydrogenase activityand poly hydroxacid synthase activity. Such cells can be used to producepolymerized acrylate.

In another example, the cells also include alanine dehydrogenase orpyruvate/glutamate transaminase activity, CoA transferase or CoAsynthetase activity, beta-alanyl-CoA ammonia lyase activity, glutamatedehydrogenase activity, and lipase or esterase activity, wherein thecells can be used to produce an ester of acrylate, such as methylacrylate, ethyl acrylate, propyl acrylate, or butyl acrylate.

Alternatively, such cells also include alanine dehydrogenase orpyruvate-glutamate transaminase activity, CoA transferase or CoAsynthetase activity, beta-alanyl-CoA ammonia lyase activity, 3-HP—CoAdehydratase activity, glutamate dehydrogenase activity,3-hydroxypropionyl-CoA hydrolase or 3-hydroxyisobutryl-CoA hydrolaseactivity, and aldehyde or alcohol dehydrogenase activity. Such cells canbe used to produce 1,3-propanediol.

In one example, the cells also have alpha-ketopantoatehydroxymethyltransferase (E.C. 2.1.2.11), alpha-ketopantoate reductase(E.C. 1.1.1.169), and pantothenate synthase (E.C. 6.3.2.1) activity.Such cells can be used to produce pantothenate. Alternatively or inaddition, the cells also have pantothenate kinase (E.C. 2.7.1.33),4′-phosphopantethenoyl-1-cysteine synthetase (E.C. 6.3.2.5),4′-phosphopantothenoylcysteine decarboxylase (E.C. 4.1.1.36),ATP:4′-phosphopantetheine adenyltransferase (E.C. 2.7.7.3), anddephospho-CoA kinase (E.C. 2.7.1.24) activity. Such cells can be used toproduce coenzyme A (CoA).

Methods to Identify Cells Having Alanine 2,3-Aminomutase Activity

A method of identifying a cell having alanine 2,3-aminomutase activityis disclosed. The method includes culturing a cell, such as aprokaryotic cell, which is functionally deleted for panD, in media whichincludes alpha-alanine, but not beta-alanine or pantothenate, or inmedia in which the cell can produce alpha-alanine from media sources ofcarbon, oxygen, hydrogen, and nitrogen, but which does not includebeta-alanine or pantothenate, and identifying cells capable of growingin the beta-alanine or pantothenate deficient-media. In particularexamples, the cell is also functionally deleted for panF. Growth of thecell indicates that the cell is producing beta-alanine fromalpha-alanine, which indicates the cell has alanine 2,3-aminomutaseactivity. In contrast, if a cell does not grow and/or survive on thebeta-alanine or pantothenate deficient-media, this indicates that thecell is not producing beta-alanine from alpha-alanine, which indicatesthe cell does not have alanine 2,3-aminomutase activity.

In one example, the cell functionally deleted for panD is transformedwith one or more mutated aminomutases, such as libraries includingmutated lysine 2,3-aminomutase, mutated leucine 2,3-aminomutase, and/ormutated lysine 5,6-aminomutase. In a particular example, the cell istransformed with a library of mutated lysine 2,3-aminomutases, prior toculturing and screening the cells. The enzyme lysine 2,3-aminomutase hasbeen previously cloned from Clostridium subterminale SB4 (Chirpich etal., J. Biol. Chem. 245:1778-89, 1970) and Bacillus subtilis (Chen etal., Biochem. J. 348:539-49, 2000), and has been shown to catalyze theinterconversion of lysine and beta-lysine. Mutant aminomutases, such asa mutant lysine 2,3-aminomutase, can be screened for their ability toconfer alanine 2,3-aminomutase activity. In addition, although apolypeptide having alanine 2,3-aminomutase activity has not beenpreviously described, such an enzyme may exist in nature. Thus, a cellfunctionally deleted for panD can be transformed with a libraryincluding a gene encoding for alanine 2,3-aminomutase, and the geneisolated by its ability to confer growth to this cell in mediacontaining alpha-alanine, or carbon, oxygen, hydrogen, and nitrogensources such that the cell can generate alpha-alanine, but notcontaining beta-alanine or pantothenate.

In another example, the method further includes identifying a mutationin the mutated aminomutase(s) following identifying a cell which growsin the media, wherein the mutated aminomutase(s) confers alanine2,3-aminomutase activity to the cell. To identify the mutation, theaminomutase nucleic acid or amino acid can be sequenced and compared toa non-mutated aminomutase sequence, to identify mutations that conferalanine 2,3-aminomutase activity to the cell.

Methods of Producing a Peptide Having Alanine 2,3-Aminomutase Activity

A method for producing alanine 2,3-aminomutase peptides having alanine2,3-aminomutase activity, is disclosed. The method includes culturingthe disclosed cells having alanine 2,3-aminomutase activity underconditions that allow the cell to produce the alanine 2,3-aminomutasepeptide. In one example, the method includes culturing cells having oneor more exogenous nucleic acid molecules which encode for an alanine2,3-aminomutase (such as a sequence which includes SEQ ID NO: 20 and/or29 or variants, fusions, or fragments thereof that retain alanine2,3-aminomutase activity), such that the alanine 2,3-aminomutase isproduced.

A method for making beta-alanine from alpha-alanine is also disclosed.In one example, the method includes culturing the disclosed cells havingalanine 2,3-aminomutase activity under conditions that allow the cell toproduce beta-alanine from alpha-alanine. In one example, the methodincludes culturing cells having one or more exogenous nucleic acidmolecules which encode for an alanine 2,3-aminomutase, such that thealanine 2,3-aminomutase is capable of producing beta-alanine fromalpha-alanine. In one example, the exogenous nucleic acid is a sequencethat includes SEQ ID NO: 20 and/or 29 or variants, fusions, or fragmentsthereof that retain alanine 2,3-aminomutase activity.

In particular examples, the cell is functionally deleted for panD, orpanD and panF.

Pathways for Producing 3-HP, Pantothenate and Derivatives Thereof

Methods and materials related to producing beta-alanine fromalpha-alanine, via an alanine 2,3-aminomutase, such as using thedisclosed alanine 2,3-aminomutase sequences and the disclosed cellshaving alanine 2,3-aminomutase activity are disclosed. In addition,methods and materials related to producing pantothenate and 3-HP frombeta-alanine, as well as CoA and organic compounds such as1,3-propanediol, acrylic acid, polymerized acrylate, esters of acrylate,polymerized 3-HP, co-polymers of 3-HP and other compounds such asbutyrates, valerates and other compounds, and esters of 3-HP, aredisclosed. Specifically, the disclosure provides alanine 2,3-aminomutasenucleic acids (such as SEQ ID NO: 20 and 29), polypeptides (such as SEQID NO: 21 and 30), host cells, and methods and materials for producingbeta-alanine from alpha-alanine, which can be used to more efficientlymake beta-alanine pantothenate and 3-HP as well as derivatives thereofsuch as CoA and organic compounds such as 1,3-propanediol, acrylic acid,polymerized acrylate, esters of acrylate, polymerized 3-HP, and estersof 3-HP.

Several metabolic pathways can be used to produce organic compounds frombeta-alanine which has been produced from alpha-alanine (FIGS. 1 and 3).

Pathways of 3-HP and It Derivatives

As shown in FIG. 1, beta-alanine can be converted into beta-alanyl-CoAthrough the use of a polypeptide having CoA transferase activity (EC2.8.3.1) or CoA synthase activity (E.C. 6.2.1.−). Beta-alanine can beproduced from alpha-alanine by endogenous polypeptides in a host cellwhich converts alpha-alanine to beta-alanine, and/or by using a celltransformed with recombinant alanine 2,3-aminomutase, such as a sequenceincluding SEQ ID NO: 20 and/or 29, or fragments, variants, or fusionsthereof that retain alanine 2,3-aminomutase activity. Beta-alanyl-CoAcan then be converted into acrylyl-CoA through the use of a polypeptidehaving beta-alanyl-CoA ammonia lyase activity (EC4.3.1.6). Acrylyl-CoAcan then be converted into 3-hydroxypropionyl-CoA (3-HP—CoA) through theuse of a polypeptide having 3-HP—CoA dehydratase activity (EC 4.2.1.−).3-HP—CoA can then be converted into 3-HP through several enzymes,including, but not limited to: a polypeptide having CoA transferaseactivity (EC 2.8.3.1), a polypeptide having 3-hydroxypropionyl-CoAhydrolase activity (EC 3.1.2.−), and a polypeptide having3-hydroxyisobutryl-CoA hydrolase activity (EC 3.1.2.4) can be used toconvert 3-HP—CoA into 3-HP.

As shown in FIG. 1, 3-HP can be made from beta-alanine by use of apolypeptide having 4-aminobutyrate and/or beta-alanine-2-oxoglutarateaminotransferase activity which generates malonic semialdehyde frombeta-alanine. The malonic semialdehyde can be converted into 3-HP with apolypeptide having 3-HP dehydrogenase activity (EC 1.1.1.59) or apolypeptide having 3-hydroxyisobutyrate dehydrogenase activity (EC1.1.1.31).

Derivatives of 3-HP can be made from beta-alanine as shown in FIG. 1.The resulting 3-HP—CoA can be converted into polymerized 3-HP by apolypeptide having poly hydroxyacid synthase activity (EC 2.3.1.−).Alternatively or in addition, 3-HP—CoA can be converted into1,3-propanediol by polypeptides having oxidoreductase activity orreductase activity.

The resulting acrylyl-CoA can be converted into polymerized acrylate bya polypeptide having poly hydroxyacid synthase activity (EC 2.3.1.−).Alternatively or in addition, acrylyl-CoA can be converted into acrylateby a polypeptide having CoA transferase activity and/or CoA hydrolaseactivity; and the resulting acrylate can be converted into an ester ofacrylate by a polypeptide having lipase or esterase activity.

The resulting 3-HP can be converted into an ester of 3-HP by apolypeptide having lipase or esterase activity (EC 3.1.1.−).Alternatively or in addition, 1,3-propanediol can be created from 3-HP,by a combination of a polypeptide having aldehyde dehydrogenase activityand a polypeptide having alcohol dehydrogenase activity.

Pathways of Pantothenate and It Derivatives

As shown in FIG. 3, pantothenate can be made from beta-alanine by apeptide having alpha-ketopantoate hydroxymethyltransferase (E.C.2.1.2.11), alpha-ketopantoate reductase (E.C. 1.1.1.169), andpantothenate synthase (E.C. 6.3.2.1) activities, which convertsbeta-alanine to pantothenate.

Derivatives of pantothenate can be made from beta-alanine as follows.The resulting pantothenate can be converted into CoA by polypeptideshaving pantothenate kinase (E.C. 2.7.1.33),4′-phosphopantethenoyl-1-cysteine synthetase (E.C. 6.3.2.5),4′-phosphopantothenoylcysteine decarboxylase (E.C. 4.1.1.36),ATP:4′-phosphopantetheine adenyltransferase (E.C. 2.7.7.3), anddephospho-CoA kinase (E.C. 2.7.1.24) activities.

Enzymes

Polypeptides having lysine 2,3-aminomutase activity as well as nucleicacid encoding such polypeptides can be obtained from various speciesincluding, but not limited to: Clostridium subterminale, E. coli, B.subtilis, Deinococcus radiodurans, Porphyromonas gingivalis, Aquifexaeolicus, or Haemophilus influenza. For example, amino acid sequenceshaving lysine 2,3-aminomutase activity are shown in SEQ ID NO: 31 for B.subtilis and in SEQ ID NO: 28 for P. gingivalis.

In another example, a nucleic acid that encodes a polypeptide havingalanine 2,3-aminomutase activity is shown in SEQ ID NO: 20 for B.subtilis (the corresponding amino acid sequence is shown in SEQ ID NO:21), and in SEQ ID NO: 29 for P. gingivalis (the corresponding aminoacid sequence is shown in SEQ ID NO: 30). In addition, otherpolypeptides having alanine 2,3-aminomutase activity as well as nucleicacids encoding such polypeptides, can be obtained using the methodsdescribed herein. For example, alanine 2,3-aminomutase variants can beused to encode a polypeptide having alanine 2,3-aminomutase activity asdescribed above.

Polypeptides having CoA transferase activity as well as nucleic acidencoding such polypeptides can be obtained from various speciesincluding, but not limited to, Megasphaera elsdenii, Clostridiumpropionicum, Clostridium kluyveri, and E. coli. For example, nucleicacid that encodes a polypeptide having CoA transferase activity is shownin SEQ ID NO: 24 for M. elsdenii. In addition, polypeptides having CoAtransferase activity (SEQ ID NO: 25) as well as nucleic acid encodingsuch polypeptides (SEQ ID NO: 24) can be obtained as described herein.For example, CoA transferase variants can be used to encode apolypeptide having CoA transferase activity. For example, the followingvariations can be made to the CoA transferase nucleic acid sequence (SEQID NO: 24): the “a” at position 49 can be substituted with an “c”; the“a” at position 590 can be substituted with a “atgg”; an “aaac” can beinserted before the “g” at position 393; or the “gaa” at position 736can be deleted. It will be appreciated that the sequences set forth inthe sequence listing can contain any number of variations as well as anycombination of types of variations, as long as the peptide retains CoAtransferase activity. In addition, the following variations can be madeto the CoA transferase amino acid sequence shown in SEQ ID NO: 25: the“k” at position 17 of can be substituted with a “p” or “h”; and the “v”at position 125 can be substituted with an “i” or “f.”

Polypeptides having beta-alanyl-CoA ammonia lyase activity as well asnucleic acid encoding such polypeptides can be obtained from variousspecies including, without limitation, C. propionicum. For example,nucleic acid encoding a polypeptide complex having beta-alanyl-CoAammonia lyase activity can be obtained from C. propionicum as describedin EXAMPLE 10. The nucleic acid encoding a beta-alanyl-CoA ammonia lyasecan contain a sequence as set forth in SEQ ID NO: 22. In addition,polypeptides having beta-alanyl-CoA ammonia lyase activity (SEQ ID NO:23) as well as nucleic acid encoding such polypeptides (SEQ ID NO: 22)can be obtained as described herein. For example, the variations to thebeta-alanyl-CoA ammonia lyase sequence shown in SEQ ID NO: 22 can beused to encode a polypeptide having beta-alanyl-CoA ammonia lyaseactivity.

Polypeptides having 3-hydroxypropionyl-CoA dehydratase activity (alsoreferred to as acrylyl-CoA hydratase activity) as well as nucleic acidencoding such polypeptides can be obtained from various speciesincluding, but not limited to, Chloroflexus aurantiacus, Candida rugosa,Rhodosprillium rubrum, and Rhodobacter capsulates. For example, anucleic acid that encodes a polypeptide having 3-hydroxypropionyl-CoAdehydratase activity is disclosed in WO 02/42418.

Polypeptides having glutamate dehydrogenase activity as well as nucleicacid encoding such polypeptides can be obtained from various species.

Polypeptides having 3-hydroxypropionyl-CoA or 3-hydroxyisobutryl-CoAhydrolase activity, as well as nucleic acid encoding such polypeptides,can be obtained from various species including, without limitation,Pseudomonas fluorescens, Rattus rattus, and Homo sapiens. For example,nucleic acid that encodes a polypeptide having 3-hydroxyisobutyryl-CoAhydrolase activity can be obtained from H. sapiens and can have asequence as set forth in GenBank accession number U66669.

Polypeptides having 4-aminobutyrate and/or beta-alanine-2-oxoglutarateaminotransferase activity, 3-HP dehydrogenase activity, and3-hydroxyisobutyrate dehydrogenase activity, as well as nucleic acidencoding such polypeptides can be obtained from various species.

Polypeptides having poly hydroxyacid synthase activity as well asnucleic acid encoding such polypeptides can be obtained from variousspecies including, without limitation, Rhodobacter sphaeroides,Comamonas acidororans, Ralstonia eutropha, and Pseudomonas oleovorans.For example, nucleic acid that encodes a polypeptide having polyhydroxyacid synthase activity can be obtained from R. sphaeroides andcan have a sequence as set forth in GenBank accession number X97200.Addition information about poly hydroxyacid synthase can be found inSong et al. (Biomacromolecules 1:433-9, 2000).

Polypeptides having acetylating aldehyde:NAD(+) oxidoreductase activity(EC 1.2.1.10) as well as nucleic acid encoding such polypeptides can beobtained from various species including, without limitation, E. coli.For example, nucleic acid that encodes a polypeptide having acylatingaldehyde dehydrogenase activity can be obtained from E. coli and canhave a sequence as set forth in GenBank accession number Y09555.

Aldehyde:NAD(+) oxidoreductase activity and alcohol:NAD(+)oxidoreductase activities can be carried out by two differentpolypeptides as described above, or carried out by a single polypeptide,such as a multi-functional aldehyde-alcohol dehydrogenase (EC 1.2.1.10)from E. coli (Goodlove et al. Gene 85:209-14, 1989; GenBank AccessionNo. M33504).

Polypeptides having aldehyde dehydrogenase (NAD(P)+) (EC 1.2.1.−)activity as well as nucleic acid encoding such polypeptides can beobtained from various species including, without limitation, S.cerevisiae. For example, nucleic acid that encodes a polypeptide havingaldehyde dehydrogenase activity can be obtained from S. cerevisiae andcan have a sequence as set forth in GenBank Accession No. Z75282(Tessier et al. FEMS Microbiol. Lett. 164:29-34, 1998).

Polypeptides having alcohol dehydrogenase activity (EC 1.1.1.1) as wellas nucleic acid encoding such polypeptides can be obtained from variousspecies including, without limitation, Z. mobilis. For example, nucleicacid that encodes a polypeptide having alcohol dehydrogenase activitycan be obtained from Z. mobilis and can have a sequence as set forth inGenBank accession No. M32100.

Polypeptides having lipase activity as well as nucleic acid encodingsuch polypeptides can be obtained from various species including,without limitation, Candida rugosa, Candida tropicalis, and Candidaalbicans. For example, nucleic acid that encodes a polypeptide havinglipase activity can be obtained from C. rugosa and can have a sequenceas set forth in GenBank accession number A81171.

Polypeptides having alpha-ketopantoate hydroxymethyltransferase andpantothenate synthase activity as well as nucleic acid encoding suchpolypeptides can be obtained from various species including, withoutlimitation, E. coli. For example, nucleic acids that encodespolypeptides having alpha-ketopantoate hydroxymethyltransferase andpantothenate synthase activity can be obtained from E. coli and can havea sequence as set forth in GenBank accession number L17086.

Polypeptides having alpha-ketopantoate reductase, pantothenate kinase,4′-phosphopantethenoyl-1-cysteine synthetase,4′-phosphopantothenoylcysteine decarboxylase, ATP:4′-phosphopantetheineadenyltransferase, and dephospho-CoA kinase activity as well as nucleicacid encoding such polypeptides can be obtained from various speciesincluding, without limitation, E. coli. For example, nucleic acids thatencodes polypeptides having alpha-ketopantoate reductase pantothenatekinase, 4′-phosphopantethenoyl-1-cysteine synthetase,4′-phosphopantothenoylcysteine decarboxylase, ATP:4′-phosphopantetheineadenyltransferase, and dephospho-CoA kinase activity can be obtainedfrom E. coli and can have a sequence as set forth in GenBank accessionnumber NC000913.

The term “polypeptide having enzymatic activity” refers to anypolypeptide that catalyzes a chemical reaction of other substanceswithout itself being destroyed or altered upon completion of thereaction. Typically, a polypeptide having enzymatic activity catalyzesthe formation of one or more products from one or more substrates. Suchpolypeptides can have any type of enzymatic activity including, withoutlimitation, the enzymatic activity or enzymatic activities associatedwith enzymes such as alanine 2,3-aminomutase, dehydratases/hydratases,3-hydroxypropionyl-CoA dehydratases/hydratases, alanine dehydrogenase,CoA transferases, 3-hydroxypropionyl-CoA hydrolases,3-hydroxyisobutryl-CoA hydrolases, CoA hydrolases, poly hydroxyacidsynthases, beta-alanine ammonia lyases, 4-aminobutyrate orbeta-alanine-2-oxoglutarate aminotransferases, 3-HP dehydrogenases,3-hydroxyisobutyrate dehydrogenases, glutamate dehydrogenases, lipases,esterases, acetylating aldehyde:NAD(+) oxidoreductases, alcohol:NAD(+)oxidoreductases, aldehyde dehydrogenases, alcohol dehydrogenaseshydroxymethyltransferases, reductases, synthases, kinases, synthetases,decarboxylases, alpha-ketopantoate hydroxymethyltransferases,alpha-ketopantoate reductases, pantothenate synthases, pantothenatekinases, 4′-phosphopantethenoyl-1-cysteine synthetase,4′-phosphopantothenoylcysteine decarboxylases, ATP:4′-phosphopantetheineadenyltransferases, dephospho-CoA kinases, acetylating aldehyde:NAD(+)oxidoreductases, alcohol:NAD(+) oxidoreductases, aldehyde dehydrogenases(NAD(P)+), alcohol dehydrogenases and adenyltransferases.

Methods of Making 3-HP, Pantothenate, and Derivatives Thereof

Each step provided in the pathways depicted in FIGS. 1 and 3 can beperformed within a cell (in vivo) or outside a cell (in vitro, e.g., ina container or column). Additionally, the organic compound products canbe generated through a combination of in vivo synthesis and in vitrosynthesis. Moreover, the in vitro synthesis step, or steps, can be viachemical reaction or enzymatic reaction.

For example, a cell or microorganism provided herein can be used toperform the steps provided in FIGS. 1 and 3, or an extract containingpolypeptides having the indicated enzymatic activities can be used toperform the steps provided in FIGS. 1 and 3. In addition, chemicaltreatments can be used to perform the conversions provided in FIGS. 1and 3. For example, acrylyl-CoA can be converted into acrylate byhydrolysis. Other chemical treatments include, without limitation, transesterification to convert acrylate into an acrylate ester.

Expression of Polypeptides

The polypeptides described herein, such as the enzymes listed in FIG. 1,can be produced individually in a host cell or in combination in a hostcell. Moreover, the polypeptides having a particular enzymatic activitycan be a polypeptide that is either naturally-occurring ornon-naturally-occurring. A naturally-occurring polypeptide is anypolypeptide having an amino acid sequence as found in nature, includingwild-type and polymorphic polypeptides. Naturally-occurring polypeptidescan be obtained from any species including, but not limited to, animal(e.g., mammalian), plant, fungal, and bacterial species. Anon-naturally-occurring polypeptide is any polypeptide having an aminoacid sequence that is not found in nature. Thus, anon-naturally-occurring polypeptide can be a mutated version of anaturally-occurring polypeptide, or an engineered polypeptide. Forexample, a non-naturally-occurring polypeptide having alanine2,3-aminomutase activity can be a mutated version of anaturally-occurring polypeptide having lysine 2,3-aminomutase activitythat has at least some alanine 2,3-aminomutase activity (such as SEQ IDNO: 21 and/or 30). A polypeptide can be mutated by, for example,sequence additions, deletions, substitutions, or combinations thereof.

Genetically modified cells are disclosed which can be used to performone or more steps of the steps in the pathways described herein or thegenetically modified cells can be used to produce the disclosedpolypeptides for subsequent use in vitro. For example, an individualmicroorganism can contain exogenous nucleic acid(s) encoding each of thepolypeptides necessary to perform the steps depicted in FIGS. 1 and 3.Such cells can contain any number of exogenous nucleic acid molecules.For example, a particular cell can contain one, two, three, or fourdifferent exogenous nucleic acid molecules with each one encoding thepolypeptide(s) necessary to convert pyruvate into 3-HP as shown in FIG.1, or a particular cell can endogenously produce polypeptides necessaryto convert pyruvate into acrylyl-CoA while containing exogenous nucleicacid that encodes polypeptides necessary to convert acrylyl-CoA into3-HP.

In addition, a single exogenous nucleic acid molecule can encode one, ormore than one, polypeptide. For example, a single exogenous nucleic acidmolecule can contain sequences that encode two, three, or even fourdifferent polypeptides. Further, the cells described herein can containa single copy, or multiple copies (e.g., about 5, 10, 20, 35, 50, 75,100 or 150 copies), of a particular exogenous nucleic acid molecule,such as a particular enzyme. The cells described herein can contain morethan one particular exogenous nucleic acid. For example, a particularcell can contain about 50 copies of exogenous nucleic acid molecule X aswell as about 75 copies of exogenous nucleic acid molecule Y.

In another example, a cell can contain an exogenous nucleic acidmolecule that encodes a polypeptide having alanine 2,3-aminomutaseactivity, for example SEQ ID NO: 20 and/or 29 (or variants, fragments,or fusions thereof that retain alanine 2,3-aminomutase activity). Suchcells can have any detectable level of alanine 2,3-aminomutase activity,including activity detected by the production of metabolites ofbeta-alanine, such as pantothenate. For example, a cell containing anexogenous nucleic acid molecule that encodes a polypeptide havingalanine 2,3-aminomutase activity can have alanine 2,3-aminomutaseactivity with a specific activity greater than about 1 μg beta-alanineformed per gram dry cell weight per hour (e.g., greater than about 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400,500, or more μg beta-alanine formed per gram dry cell weight per hour).Alternatively, a cell can have alanine 2,3-aminomutase activity suchthat a cell extract from 1×10⁶ cells has a specific activity greaterthan about 1 ng beta-alanine formed per mg total protein per minute(e.g., greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125,150, 200, 250, 300, 350, 400, 500, or more ng beta-alanine formed per mgtotal protein per minute).

A nucleic acid molecule encoding a polypeptide having enzymatic activitycan be identified and obtained using any method such as those describedherein. For example, nucleic acid molecules that encode a polypeptidehaving enzymatic activity can be identified and obtained using commonmolecular cloning or chemical nucleic acid synthesis procedures andtechniques, including PCR. In addition, standard nucleic acid sequencingtechniques and software programs that translate nucleic acid sequencesinto amino acid sequences based on the genetic code can be used todetermine whether or not a particular nucleic acid has any sequencehomology with known enzymatic polypeptides. Sequence alignment softwaresuch as MEGALIGN (DNASTAR, Madison, Wis., 1997) can be used to comparevarious sequences.

In addition, nucleic acid molecules encoding known enzymaticpolypeptides can be mutated using common molecular cloning techniques(e.g., site-directed mutagenesis). Possible mutations include, withoutlimitation, deletions, insertions, and base substitutions, as well ascombinations of deletions, insertions, and base substitutions. Further,nucleic acid and amino acid databases (e.g., GenBank) can be used toidentify a nucleic acid sequence that encodes a polypeptide havingenzymatic activity. Briefly, any amino acid sequence having somehomology to a polypeptide having enzymatic activity, or any nucleic acidsequence having some homology to a sequence encoding a polypeptidehaving enzymatic activity can be used as a query to search GenBank. Theidentified polypeptides then can be analyzed to determine whether or notthey exhibit enzymatic activity.

In addition, nucleic acid hybridization techniques can be used toidentify and obtain a nucleic acid molecule that encodes a polypeptidehaving enzymatic activity. Briefly, any nucleic acid molecule thatencodes a known enzymatic polypeptide, or fragment thereof, can be usedas a probe to identify a similar nucleic acid molecules by hybridizationunder conditions of moderate to high stringency. Such similar nucleicacid molecules then can be isolated, sequenced, and analyzed todetermine whether the encoded polypeptide has enzymatic activity.

Expression cloning techniques also can be used to identify and obtain anucleic acid molecule that encodes a polypeptide having enzymaticactivity. For example, a substrate known to interact with a particularenzymatic polypeptide can be used to screen a phage display librarycontaining that enzymatic polypeptide. Phage display libraries can begenerated as described (Burritt et al., Anal. Biochem. 238:1-13, 1990),or can be obtained from commercial suppliers such as Novagen (Madison,Wis.).

Further, polypeptide sequencing techniques can be used to identify andobtain a nucleic acid molecule that encodes a polypeptide havingenzymatic activity. For example, a purified polypeptide can be separatedby gel electrophoresis, and its amino acid sequence determined by, forexample, amino acid microsequencing techniques. Once determined, theamino acid sequence can be used to design degenerate oligonucleotideprimers. Degenerate oligonucleotide primers can be used to obtain thenucleic acid encoding the polypeptide by PCR. Once obtained, the nucleicacid can be sequenced, cloned into an appropriate expression vector, andintroduced into a microorganism.

Any method can be used to introduce an exogenous nucleic acid moleculeinto a cell. For example, heat shock, lipofection, electroporation,conjugation, fusion of protoplasts, and biolistic delivery are commonmethods for introducing nucleic acid into bacteria and yeast cells.(See, e.g., Ito et al., J. Bacterol. 153:163-8, 1983; Durrens et al.,Curr. Genet. 18:7-12, 1990; Sambrook et al., Molecular cloning: Alaboratory manual, Cold Spring Harbour Laboratory Press, New York, USA,second edition, 1989; and Becker and Guarente, Methods in Enzymology194:182-7, 1991). Other methods for expressing an amino acid sequencefrom an exogenous nucleic acid molecule include, but are not limited to,constructing a nucleic acid such that a regulatory element promotes theexpression of a nucleic acid sequence that encodes a polypeptide.Typically, regulatory elements are DNA sequences that regulate theexpression of other DNA sequences at the level of transcription. Thus,regulatory elements include, without limitation, promoters, enhancers,and the like. Any type of promoter can be used to express an amino acidsequence from an exogenous nucleic acid molecule. Examples of promotersinclude, without limitation, constitutive promoters, tissue-specificpromoters, and promoters responsive or unresponsive to a particularstimulus (e.g., light, oxygen, chemical concentration). Methods fortransferring nucleic acids into mammalian cells are also known, such asusing viral vectors.

An exogenous nucleic acid molecule contained within a particular cell ofthe disclosure can be maintained within that cell in any form. Forexample, exogenous nucleic acid molecules can be integrated into thegenome of the cell or maintained in an episomal state. That is, a cellcan be a stable or transient transformant. A microorganism can containsingle or multiple copies (e.g., about 5, 10, 20, 35, 50, 75, 100 or 150copies), of a particular exogenous nucleic acid molecule, such as anucleic acid encoding an enzyme.

Production of Organic Acids and Related Products Via Host Cells

The nucleic acid and amino acid sequences provided herein can be usedwith cells to produce beta-alanine, pantothenate and 3-HP, as well asderivatives thereof such as CoA, and organic compounds such as1,3-propanediol, acrylic acid, polymerized acrylate, esters of acrylate,esters of 3-HP, and polymerized 3-HP. Such cells can be from anyspecies, such as those listed within the taxonomy web pages at theNational Institutes of Health. The cells can be eukaryotic orprokaryotic. For example, genetically modified cells can be mammaliancells (e.g., human, murine, and bovine cells), plant cells (e.g., corn,wheat, rice, and soybean cells), fungal cells (e.g., Aspergillus andRhizopus cells), yeast cells, or bacterial cells (e.g., Lactobacillus,Lactococcus, Bacillus, Escherichia, and Clostridium cells). In oneexample, a cell is a microorganism. The term “microorganism” refers toany microscopic organism including, but not limited to, bacteria, algae,fungi, and protozoa. Thus, E. coli, B. subtilis, B. licheniformis, S.cerevisiae, Kluveromyces lactis, Candida blankii, Candida rugosa, andPichia pastoris are microorganisms and can be used as described herein.In another example, the cell is part of a larger organism, such as aplant, such as a transgenic plant. Examples of plants that can be usedto make 3-HP, pantothenate, or other organic compounds from beta-alanineinclude, but are not limited to, genetically engineered plant crops suchas corn, rice, wheat, and soybean.

In one example, a cell is genetically modified such that a particularorganic compound is produced. In one embodiment, cells make 3-HP and/orpantothenate from beta-alanine, such as the pathways shown in FIGS. 1and 3. In another embodiment, the cells make derivatives of 3-HP and/orpantothenate, such as CoA, and organic compounds such as1,3-propanediol, acrylic acid, polymerized acrylate, esters of acrylate,esters of 3-HP, and polymerized 3-HP.

In one example, cells that are genetically modified to synthesize aparticular organic compound contain one or more exogenous nucleic acidmolecules that encode polypeptides having specific enzymatic activities.For example, a microorganism can contain exogenous nucleic acid thatencodes a polypeptide having 3-hydroxypropionyl-CoA dehydrataseactivity. In this case, acrylyl-CoA can be converted into3-hydroxypropionic acid-CoA which can lead to the production of 3-HP. Acell can be given an exogenous nucleic acid molecule that encodes apolypeptide having an enzymatic activity that catalyzes the productionof a compound not normally produced by that cell. Alternatively, a cellcan be given an exogenous nucleic acid molecule that encodes apolypeptide having an enzymatic activity that catalyzes the productionof a compound that is normally produced by that cell. In this case, thegenetically modified cell can produce more of the compound, or canproduce the compound more efficiently, than a similar cell not havingthe genetic modification.

In another example, a cell containing an exogenous nucleic acid moleculethat encodes a polypeptide having enzymatic activity that leads to theformation of 3-HP, pantothenate, and/or derivatives thereof, isdisclosed. The produced product(s) can be secreted from the cell,eliminating the need to disrupt cell membranes to retrieve the organiccompound. In one example, the cell produces 3-HP, pantothenate, and/orderivatives thereof, with the concentration of the product(s) being atleast about 100 mg per L (e.g., at least about 1 g/L, 5 g/L, 10 g/L, 25g/L, 50 g/L, 75 g/L, 80 g/L, 90 g/L, 100 g/L, or 120 g/L). Whendetermining the yield of a compound such as 3-HP, pantothenate, and/orderivatives thereof for a particular cell, any method can be used. See,e.g., Applied Environmental Microbiology 59(12):4261-5 (1993). A cellwithin the scope of the disclosure can utilize a variety of carbonsources.

A cell can contain one or more exogenous nucleic acid molecules thatencodes a polypeptide(s) having enzymatic activity that leads to theformation of 3-HP, pantothenate, and/or derivatives thereof, such asCoA, 1,3-propanediol, acrylic acid, poly-acrylate, acrylate-esters,3-HP-esters, and polymers and copolymers containing 3-HP. Methods ofidentifying cells that contain exogenous nucleic acid(s) are well known.Such methods include, without limitation, PCR and nucleic acidhybridization techniques such as Northern and Southern analysis (seehybridization described herein). In some cases, immunohisto-chemical andbiochemical techniques can be used to determine if a cell containsparticular nucleic acid(s) by detecting the expression of thepolypeptide(s) encoded by that particular nucleic acid molecule(s). Forexample, an antibody having specificity for a polypeptide can be used todetermine whether or not a particular cell contains nucleic acidencoding that polypeptide. Further, biochemical techniques can be usedto determine if a cell contains a particular nucleic acid moleculeencoding a polypeptide having enzymatic activity by detecting an organicproduct produced as a result of the expression of the polypeptide havingenzymatic activity. For example, detection of 3-HP after introduction ofexogenous nucleic acid that encodes a polypeptide having3-hydroxypropionyl-CoA dehydratase activity into a cell that does notnormally express such a polypeptide can indicate that the cell not onlycontains the introduced exogenous nucleic acid molecule but alsoexpresses the encoded polypeptide from that introduced exogenous nucleicacid molecule. Methods for detecting specific enzymatic activities orthe presence of particular organic products are well known, for example,the presence of an organic compound such as 3-HP can be determined asdescribed in Sullivan and Clarke (J. Assoc. Offic. Agr. Chemists,38:514-8, 1955).

Cells with Reduced Polypeptide Activity

Genetically modified cells having reduced polypeptide activity aredisclosed. The term “reduced” or “decreased” as used herein with respectto a cell and a particular polypeptide's activity refers to a lowerlevel of activity than that measured in a comparable cell of the samespecies. For example, a particular microorganism lacking enzymaticactivity X has reduced enzymatic activity X if a comparablemicroorganism has at least some enzymatic activity X.

A cell can have the activity of any type of polypeptide reducedincluding, without limitation, enzymes, transcription factors,transporters, receptors, signal molecules, and the like. For example, acell can contain an exogenous nucleic acid molecule that disrupts aregulatory and/or coding sequence of a polypeptide having panD activity.Disrupting panD can prevent a cell from making beta-alanine.

Reduced polypeptide activities can be the result of lower polypeptideconcentration, lower specific activity of a polypeptide, or combinationsthereof. Many different methods can be used to make a cell havingreduced polypeptide activity. For example, a cell can be engineered tohave a disrupted regulatory sequence or polypeptide-encoding sequenceusing common mutagenesis or knock-out technology. (Methods in YeastGenetics (1997 edition), Adams, Gottschling, Kaiser, and Sterns, ColdSpring Harbor Press, 1998; Datsenko and Wanner, Proc. Natl. Acad. Sci.USA 97: 6640-5, 2000). Alternatively, antisense technology can be usedto reduce the activity of a particular polypeptide. For example, a cellcan be engineered to contain a cDNA that encodes an antisense moleculethat prevents a polypeptide from being translated. The term “antisensemolecule” encompasses any nucleic acid molecule or nucleic acid analog(e.g., peptide nucleic acids) that contains a sequence that correspondsto the coding strand of an endogenous polypeptide. An antisense moleculealso can have flanking sequences (e.g., regulatory sequences). Thus,antisense molecules can be ribozymes or antisense oligonucleotides. Aribozyme can have any general structure including, without limitation,hairpin, hammerhead, or axhead structures, provided the molecule cleavesRNA. Further, gene silencing can be used to reduce the activity of aparticular polypeptide.

A cell having reduced activity of a polypeptide can be identified usingany method. For example, enzyme activity assays such as those describedherein can be used to identify cells having a reduced enzyme activity.

Production of Organic Acids and Related Products Via In Vitro Techniques

Purified polypeptides having enzymatic activity can be used alone or incombination with cells to produce pantothenate, 3-HP, and/or derivativesthereof such as CoA, and organic compounds such as 1,3-propanediol,acrylic acid, polymerized acrylate, esters of acrylate, esters of 3-HP,and polymerized 3-HP. For example, a preparation including asubstantially pure polypeptide having 3-hydroxypropionyl-CoA dehydrataseactivity can be used to catalyze the formation of 3-HP—CoA, a precursorto 3-HP.

Further, cell-free extracts containing a polypeptide having enzymaticactivity can be used alone or in combination with purified polypeptidesand/or cells to produce pantothenate, 3-HP, and/or deviates thereof. Forexample, a cell-free extract which includes a polypeptide having CoAtransferase activity can be used to form beta-alanyl-CoA frombeta-alanine, while a microorganism containing polypeptides which havethe enzymatic activities necessary to catalyze the reactions needed toform 3-HP from beta-alanyl-CoA can be used to produce 3-HP. In anotherexample, a cell-free extract which includes alpha-ketopantoatehydroxymethyltransferase (E.C. 2.1.2.11), alpha-ketopantoate reductase(E.C. 1.1.1.169), and pantothenate synthase (E.C. 6.3.2.1) can be usedto form pantothenate from beta-alanine. Any method can be used toproduce a cell-free extract. For example, osmotic shock, sonication,and/or a repeated freeze-thaw cycle followed by filtration and/orcentrifugation can be used to produce a cell-free extract from intactcells.

A cell, purified polypeptide, and/or cell-free extract can be used toproduce 3-HP that is, in turn, treated chemically to produce anothercompound. For example, a microorganism can be used to produce 3-HP,while a chemical process is used to modify 3-HP into a derivative suchas polymerized 3-HP or an ester of 3-HP. Likewise, a chemical processcan be used to produce a particular compound that is, in turn, convertedinto 3-HP or other organic compound (e.g., 1,3-propanediol, acrylicacid, polymerized acrylate, esters of acrylate, esters of 3-HP, andpolymerized 3-HP) using a cell, substantially pure polypeptide, and/orcell-free extract described herein. For example, a chemical process canbe used to produce acrylyl-CoA, while a microorganism can be usedconvert acrylyl-CoA into 3-HP.

Similarly, a cell, purified polypeptide, and/or cell-free extract can beused to produce pantothenate that is, in turn, treated chemically toproduce another compound. For example, a microorganism can be used toproduce pantothenate, while a chemical process is used to modifypantothenate into a derivative such as CoA. Likewise, a chemical processcan be used to produce a particular compound that is, in turn, convertedinto pantothenate or other compound (e.g., CoA) using a cell,substantially pure polypeptide, and/or cell-free extract describedherein. For example, a chemical process can be used to producepantothenate, while a microorganism can be used convert pantothenic acidinto CoA.

Fermentation of Cells to Produce Organic Acids

A method for producing pantothenate, 3-HP, and/or derivatives thereof byculturing a production cells, such as a microorganism, in culture mediumsuch that pantothenate, 3-HP, and/or derivatives thereof, is produced,is disclosed. In general, the culture media and/or culture conditionscan be such that the microorganisms grow to an adequate density andproduce the product efficiently. For large-scale production processes,any method can be used such as those described elsewhere (Manual ofIndustrial Microbiology and Biotechnology, 2^(nd) Edition, Editors:Demain and Davies, ASM Press; and Principles of Fermentation Technology,Stanbury and Whitaker, Pergamon).

Briefly, a large tank (e.g., a 100 gallon, 200 gallon, 500 gallon, ormore tank) containing appropriate culture medium with, for example, aglucose carbon source is inoculated with a particular microorganism.After inoculation, the microorganisms are incubated to allow biomass tobe produced. Once a desired biomass is reached, the broth containing themicroorganisms can be transferred to a second tank. This second tank canbe any size. For example, the second tank can be larger, smaller, or thesame size as the first tank. Typically, the second tank is larger thanthe first such that additional culture medium can be added to the brothfrom the first tank. In addition, the culture medium within this secondtank can be the same as, or different from, that used in the first tank.For example, the first tank can contain medium with xylose, while thesecond tank contains medium with glucose.

Once transferred, the microorganisms can be incubated to allow for theproduction of pantothenate, 3-HP, and/or derivatives thereof. Onceproduced, any method can be used to isolate the formed product. Forexample, common separation techniques can be used to remove the biomassfrom the broth, and common isolation procedures (e.g., extraction,distillation, and ion-exchange procedures) can be used to obtain thepantothenate, 3-HP, and/or derivatives thereof from themicroorganism-free broth. Alternatively, the product can be isolatedwhile it is being produced, or it can be isolated from the broth afterthe product production phase has been terminated.

Products Created from the Disclosed Biosynthetic Routes

The compounds produced from any of the steps provided in FIGS. 1 and 3can be chemically converted into other organic compounds. For example,3-HP can be hydrogenated to form 1,3-propanediol, a valuable polyestermonomer. Hydrogenating an organic acid such as 3-HP can be performedusing any method such as those used to hydrogenate succinic acid and/orlactic acid. For example, 3-HP can be hydrogenated using a metalcatalyst. In another example, 3-HP can be dehydrated to form acrylicacid. Any method can be used to perform a dehydration reaction. Forexample, 3-HP can be heated in the presence of a catalyst (e.g., a metalor mineral acid catalyst) to form acrylic acid. 1,3-propanediol also canbe created using polypeptides having oxidoreductase activity (e.g.,enzymes in the 1.1.1.− class of enzymes) in vitro or in vivo.

In another example, pantothenate can be used to form coenzyme A.Polypeptides having pantothenate kinase (E.C. 2.7.1.33),4′-phosphopantethenoyl-1-cysteine synthetase (E.C. 6.3.2.5),4′-phosphopantothenoylcysteine decarboxylase (E.C. 4.1.1.36),ATP:4′-phosphopantetheine adenyltransferase (E.C. 2.7.7.3), anddephospho-CoA kinase (E.C. 2.7.1.24) activities can be used to producecoenzyme A.

Production of 1,3-propanediol

Methods of producing 1,3-propanediol, and cells for such production, aredisclosed. 1,3-propanediol can be generated from either 3-HP—CoA or3-HP. Cells or microorganisms producing 3-HP—CoA or 3-HP can beengineered to make 1,3-propanediol by cloning genes which encode forenzymes having oxidoreductase/dehydrogenase type activity.

For example, 3-HP—CoA can be converted to 1,3-propanediol in thepresence of an enzyme having acetylating aldehyde:NAD(+) oxidoreductaseand alcohol:NAD(+) oxidoreductase activities. Such conversion can beperformed in vivo, in vitro, or a combination thereof. These activitiescan be carried out by a single polypeptide or by two differentpolypeptides. Single enzymes include the multi-functionalaldehyde-alcohol dehydrogenase (EC 1.2.1.10) from E. coli (Goodlove etal. Gene 85:209-14, 1989; GenBank Accession No. M33504). Enzymes havinga singular activity of acetylating aldehyde:NAD(+) oxidoreductase (EC1.2.1.10) or alcohol:NAD(+) oxidoreductase (EC 1.1.1.1) have beendescribed. Genes encoding for acylating aldehyde dehydrogenase from E.coli (GenBank Accession No. Y09555) and alcohol dehydrogenase from Z.mobilis (GenBank Accession No. M32100) have been isolated and sequenced.The genes encoding for these enzymes can be cloned into a 3-HP—CoAproducing organism or cell by well-known molecular biology techniques.Expression of these enzymes in 3-HP—CoA producing organisms or cellswill impart it the ability to convert 3-HP—CoA to 1,3-propanediol. Thesubstrate specificity of these enzymes for 3-HP—CoA can be changed orimproved using well-known techniques such as error prone PCR or mutatorE. coli strains.

Conversion of 3-HP to 1,3-propanediol can be achieved by contacting 3-HPwith enzymes having aldehyde dehydrogenase (NAD(P)+) (EC 1.2.1.−) andalcohol dehydrogenase (EC 1.1.1.1) activity. Such conversion can beperformed in vivo, in vitro, or a combination thereof. For example,cloning and expressing these genes in a 3-HP producing microorganism orcell will impart the ability of the cell or organism to convert 3-HP to1,3-propanediol. The substrate specificity of these enzymes for 3-HP—CoAcan be changed or improved using well-known techniques as describedabove.

The formation of 1,3-propanediol during fermentation or in an in vitroassay can be analyzed using a High Performance Liquid Chromatography(HPLC). The chromatographic separation can be achieved by using aBio-Rad 87H ion-exchange column. A mobile phase of 0.0 IN sulfuric acidis passed at a flow rate of 0.6 ml/min and the column maintained at atemperature of 45-65° C. The presence of 1,3-propanediol in the samplecan be detected using a refractive index detector (Skraly et al., Appl.Environ. Microbiol. 64:98-105, 1998).

Example 1 Cloning a Bacillus subtilis Lysine 2,3-Aminomutase (KAM Gene)

To identify an alanine 2,3-aminomutase that produces beta-alanine foralanine, enzymes which carry out similar reactions, but which do notaccept alanine or beta-alanine as substrates, were randomly mutated andthen screened to identify mutant enzymes that have alanine2,3-aminomutase activity. This example describes cloning lysine2,3-aminomutase (E.C. 5.4.3.2) from Bacillus subtilis (SEQ ID NO: 3 and31). One skilled in the art will understand that similar methods can beused to clone a lysine 2,3-aminomutase from any desired organism.

The B. subtilis lysine 2,3-aminomutase was chosen because it wasreported to be stable to air, thus permitting selection for activityunder both anaerobic and aerobic conditions. In addition, because thisenzyme has lower specific activity than the lysine 2,3-aminomutase of C.subterminale, deleterious effects to the E. coli host by overexpressionof active lysine 2,3-aminomutase or alanine 2,3-aminomutase werereduced.

To clone the B. subtilis KAM gene encoding lysine 2,3-aminomutase, thefollowing methods were used. B. subtilis ATCC 6051 was obtained fromATCC (American Type Culture Collection (ATCC), Manassas, Va.) andchromosomal DNA prepared using the Genomic Tip 20/G (Qiagen, Valencia,Calif.) following the procedure recommended by the manufacturer. Primersdesigned to amplify the KAMgene by PCR were based on the complete B.subtilis genome sequence (GenBank Accession No: NC 000964) and thesequences disclosed in Chen et al. (Biochem. J. 348:539-49, 2000) and inU.S. Pat. No. 6,248,874. The PCR primers:GCGCGAGGAGGAGTTCATATGAAAAACAAATGGTATAAAC (SEQ ID NO: 1), andCGGGCACCGCTTCGAGGC GGC CGC ACCATTCGCATG (SEQ ID NO: 2) were used, wherethe underlined nucleotides are the NdeI and NotI sites used for cloningthe PCR product into plasmids.

The PCR reaction (100 μl total volume) contained 0.5 μg B. subtilischromosomal DNA, 0.2 μM each primer (SEQ ID NOS: 1 and 2), 10 μL10×PfuTurbo reaction buffer (Stratagene, Inc., La Jolla, Calif.), 0.2 mMeach nucleotide triphosphate, and 5 units of PfuTurbo DNA polymerase(Stratagene). The PCR reaction was heated at 95° C. for 2 minutes, thensubjected to 30 cycles of 95° C. for 30 seconds, 58° C. for 30 seconds,72° C. for 2 minutes, and then held at 72° for an additional 10 minutes.

The resulting PCR product was precipitated by the addition of 3111Pellet Paint Co-Precipitant (Novagen, Inc., Madison, Wis.), 100 μl 5Mammonium acetate, and 400 μl ethanol. The resuspended reaction wasdigested with NdeI and NotI (New England Biolabs, Inc., Beverly, Mass.),purified with the QIAquick PCR Purification Kit (Qiagen), and ligatedwith the Rapid DNA Ligation Kit (Roche Molecular Biochemicals,Indianapolis, Ind.) into pET-22b(+) (Novagen) or pPRONde digested withthe same enzymes to generate the plasmids pET-KAM1 and pPRO-KAM1,respectively. Plasmid pPRONde is a derivative of pPROLar.A122 (ClontechLaboratories, Inc., Palo Alto, Calif.) in which an NdeI site wasconstructed at the intiator ATG codon by oligonucleotide-directedmutagenesis using the QuikChange Site-Directed Mutagenesis kit fromStratagene. Expression of lysine 2,3-aminomutase in these vectors isdriven by the T7 promoter in pET22(b) or a hybrid lac/ara promoter inpPRO-Nde. Ligations were transformed into E. coli DH5αc (LifeTechnologies, Gaithersburg, Md.) and clones verified by sequencing. TheB. subtilis K₄M gene is shown in SEQ ID NO: 3 (the amino acid sequenceis shown in SEQ ID NO: 31), and was mutagenized as described below, toidentify mutants having alanine 2,3-aminomutase activity.

Example 2 In Vitro Mutagenesis of a B. subtilis KAM Gene

To introduce mutations into the B. subtilis KAM gene (SEQ ID NO: 3) invitro, several error-prone PCR methods were used. Similar methods can beused to introduce mutations into any KAM gene encoding a lysine2,3-aminomutase, such as a KAM gene from Deinococcus radiodurans(GenBank Accession No: RDR02336), which is 52% identical to the B.subtilis KAMprotein sequence, Clostridium subterminale (GenBankAccession No: AF159146), or P. gingivalis (Incomplete genome, TheInstitute for Genomic Research, see EXAMPLE 5).

In one method, a GeneMorph PCR Mutagenesis Kit (Stratagene) was used asfollows. Reactions of 50 μL were set up with 10, 1, 0.1, or 0.01 ng oftemplate pET-KAM1 DNA and 125 ng each of the T7 promoter primer and theT7 terminator primer (sequences as given in the Novagen product catalog)as recommended by the manufacturer, heated at 94° C. for 30 seconds,subjected to 30 cycles of 94° C. for 30 seconds, 50° C. for 30 seconds,72° C. for 2 minutes, and then held at 72° for an additional 10 minutes.

The resulting PCR products were precipitated with 3 μl Pellet PaintCo-Precipitant (Novagen), 50 μl 5M ammonium acetate, 200 μl ethanol,resuspended, digested with NdeI and NotI, and 120 ng of each mutagenicPCR product was ligated to pPRONde digested with the same endonucleasesusing the Rapid DNA Ligation Kit (Roche Molecular Biochemicals). Theligation mixes were digested with restriction endonuclease BamHI (NewEngland Biolabs) to linearize residual vector DNA without insert,precipitated with ethanol as described, and transformed intoelectrocompetent ElectroMax DH10B E. coli cells (Invitrogen, Carlsbad,Calif.). Kanamycin-resistant transformants from each library, containing15,000-20,000 clones, were scrapped off the selection plates and themutagenized plasmid libraries prepared using Plasmid Midi Kit (Qiagen).

In a second method, based on the Mn-dITP PCR method of Xu et al.(BioTechniques 27:1102-8, 1999), an initial round of manganese-inducederror-prone PCR was conducted using pET-KAM1 DNA as template and primershomologous to the T7 promoter and T7 terminator regions. The reactionmixture (50 μL) contained 1× Taq PCR buffer with 2 mM MgCl₂, 100 ng ofDNA, 40 μM MnCl₂, 0.2 μM of each primer, 200 μM of each dNTP, and fiveunits of Taq polymerase (Roche Molecular Biochemicals). The PCR programincluded of an initial denaturation at 94° C. for 2 minutes; 20 cyclesof 94° C. for 30 seconds, 54° C. for 1 minute, and 72° C. for 2.25minutes; and a final extension at 72° C. for 7 minutes.

Three microliters of PCR product was used as template in a second roundof PCR using dITP to enhance mispairing of nucleotides duringamplification. The second-round PCR mixture (100 μL) contained 1× Taqpolymerase PCR buffer with 2 mM MgCl₂, 40 μM dITP, 0.2 μM of eachprimer, 200 μM of each dNTP, and 10 units of Taq DNA polymerase. The PCRprogram was identical to that for the first round but consisted of 30cycles. The PCR product was separated on a 1% TAE-agarose gel andpurified using a QIAquick Gel Purification procedure (Qiagen). Thepurified PCR product was digested with the restriction enzymes NdeI andNotI and ligated into pPRO-Nde vector that had been digested with thesame enzymes, gel purified, and dephosphorylated with shrimp alkalinephosphatase (Roche). The ligation reaction was conducted using T4 DNAligase (New England BioLabs) at 16° C. for 16 hours, after which anothervolume of 1× ligation buffer and ligase was added and the reactioncontinued for two hours at room temperature.

The ligation reaction was purified using a QIAquick PCR Purificationcolumn and eluted in 30 μL of water. Two microliters of the reactionwere transformed into E. coli Electromax™ DH10B™ (Life Technologies,Inc.) cells and plated on LB media containing 25 μg/mL of kanamycin.Control ligations indicated a background level (vectors with no insert)of less than 3%. Multiple transformations were done to obtainapproximately 40,000 colonies. Colonies were scrapped from plates andplasmid DNA prepared using the Qiagen MiniSpin Plasmid procedure.Plasmid DNA was precipitated with ammonium acetate and ethanol toincrease its concentration before transformation into selection hosts.Plasmid DNA was also isolated from single colonies and sequenced toobtain an estimate of the mutation rate. The average mutation rate withthis method was 1.3 altered nucleotides per Kb.

In a third method, mutagenic PCR was conducted based on the protocol ofCadwell and Joyce (PCR Methods Appl. 2:28-33, 1992). This method usedvarious dilutions of a mutagenic buffer containing 21.2 mM MgCl₂, 2.0 mMMnCl₂, 3.2 mM dTTP, and 3.2 mM dCTP. The following volumes of mutagenicbuffer were added to separate PCR reactions (each of final volume 100μl): 0, 1.56, 3.13, 6.25, 12.5, and 25 μL, in addition to 1× Taq PCRbuffer with 1.5 mM MgCl₂, 0.25 μM of each primer, 200 μM of each dNTP,50 ng of pET-KAM1 template DNA, and 10 units of Taq DNA polymerase(Roche). The PCR program included an initial denaturation at 94° C. for2 minutes; 30 cycles of 94° C. for 30 seconds, 54° C. for 1 minute, and72° C. for 2.25 minutes; and a final extension at 72° C. for 7 minutes.

Following PCR, the reactions were treated to eliminate the Taqpolymerase by adding EDTA to a final concentration of 5 mM, SDS to 0.5%,and proteinase K to 50 μg/mL (Matsumura and Ellington, Mutagenic PCR ofProtein-Coding Genes for In Vitro Evolution. Methods in MolecularBiology. Vol 182: In Vitro Mutagenesis, 2^(nd) ed. Ed. J. Braman Hamana.Press Inc. Totowa, N.J., 2001). The reactions were heated to 65° C. for15 minutes and gel purified as described above. The first fourtreatments produced sufficient PCR product for cloning. PCR product wasdigested, ligated into pPRO-Nde, and transformed into E. coliElectromax™ DH10B™ cells. Plasmid DNA was isolated from single coloniesand sequenced to obtain an estimate of the mutation rate. The averagemutation rate for treatments 1-4 varied from 0 to 0.47% (0 to 4.7altered nucleotides per Kb). Multiple transformations were conducted toobtain approximately 50,000 colonies for each selected treatment.Colonies were scrapped from plates and plasmid DNA prepared using aQiagen MiniSpin Plasmid procedure. Plasmid DNA was precipitated withammonium acetate and ethanol to increase its concentration beforetransformation into selection hosts.

Example 3 In Vivo Mutagenesis of a B. subtilis KAM Gene

To introduce mutations into the B. subtilis KAM gene (SEQ ID NO: 3) invivo, pPRO-KAM1 was passaged through the E. coli XL1Red (Stratagene)mutator strain. Approximately 50 ng of plasmid pPRO-KAM1 was transformedinto competent XL1-Red cells as directed by the manufacturer, andtransformants plated on LB medium containing 25 μg/ml kanamycin.Approximately 200 transformants selected at random were scrapped off thetransformation plates and inoculated into two portions of 5 ml LB brothcontaining 25 μg/ml kanamycin. One portion was grown overnight at 30°C., the other at 37° C.

A small aliquot of each portion was inoculated into fresh LB brothcontaining 25 μg/ml kanamycin, while mutagenized plasmid DNA wasextracted from 1.5 ml of each culture using the QiaSpin Mini kit(Qiagen). Overnight growth and plasmid DNA extraction was repeated twomore times, generating mutagenized plasmid libraries from two differenttemperatures and three cycles of increasing exposure to the mutatorstrain. The plasmid DNAs were concentrated by ethanol precipitationprior to transformation into selection strains.

Example 4 Construction of E. coli ΔpanD::CAT Strain

To identify genes encoding polypeptides that can perform the alanine2,3-aminomutase reaction, an efficient screen or selection for thedesired activity is needed. Therefore, a selection method was developedby recognizing that E. coli uses beta-alanine for the synthesis ofpantothenic acid which in turn is a component of coenzyme A (CoA) and ofacyl carrier protein (ACP). CoA and ACP are the predominant acyl groupcarriers in living organisms, and are essential for growth.

In E. coli, the primary route to beta-alanine is from aspartate in areaction catalyzed by aspartate decarboxylase (E.C. 4.1.1.1.1), which isencoded by the panD gene (FIG. 3). A functional deletion mutation ofpanD results in beta-alanine auxotrophy and growth inhibition, which canalleviated by the exogenous addition of pantothenate or beta-alanine, orby the production of beta-alanine from another source.

Two E. coli strains were used in the screen, both of which are deficientin beta-alanine synthesis. The strain DV1 (#6865, E. coli Genetic StockCenter, New Haven Conn.; Vallari and Rock, J. Bacteriol. 164:136-42,1985) is an E. coli mutant made by chemical mutagenesis, which has host(chromosomal) mutations of both the panF and panD genes which rendersboth genes non-functional. The panF gene encodes the uptake ofpantothenate from the medium, and thus the combination of panD and panFprovides a more stringent requirement for beta-alanine for growth.Therefore, although the DV1 strain was known, its use for selectingcells having alanine 2,3-aminomutase activity was not previously known.

The other selection strain, BW25113 ΔpanD::CAT, includes a deletion ofthe panD locus, to prevent revertants of the panD mutation which wouldbe able to grow without exogenous beta-alanine. This strain, which hasan insertion of a chloramphenicol resistance marker conferred by the CATgene into the panD locus, was constructed using the gene inactivationmethod of Datsenko and Wanner (Proc. Nat. Acad. Sci. USA 97: 6640-5,2000) using E. coli strains BW25113/pKD46 and BW 25141/pKD3 for the E.Coli Genetic Stock Center.

The CAT gene of pKD3 was amplified using primersTATCAATTCGTTACAGGCGATACATGGCACGCTTCGGCGCGTGTAGGCTGGAGCT GCTTC (SEQ IDNO: 4) and GATGTCGCGGCTGGTGAGTAACCAGCCGCAGGGATAACAACATATGAATATCCTC CTTAG(SEQ ID NO: 5), where the underlined sequence corresponds to the regionsin the E. coli chromosome immediately upstream and downstream of thepanD locus, respectively, and the non-underlined regions are homologousto regions in pKD3 that permit amplification of a fragment containingthe CAT gene. The PCR reaction included 30 μl 10× concentrated PCRbuffer (Roche Molecular Biochemicals), plasmid pKD3, 0.2 mM each dNTP,0.2 μM each primer, and 15 units Taq polymerase (Roche MolecularBiochemicals) in a final volume of 300 μl. The PCR reaction wasincubated at 95° C. for 30 seconds followed by 30 cycles of 95° C. for30 seconds, 45° C. for 30 seconds, 72° C. for 1 min, then 72° C. for 10min. The PCR product was precipitated with ethanol, digested with DpnI,purified with the QIAquick PCR Purification Kit (Qiagen), andtransformed into BW25113/pKD46 expressing the recombination functions.Transformants were plated on LB plates containing 25 μg/mlchloramphenicol and 5 μM beta-alanine.

Chloramphenicol-resistant transformations were single-colony purified onnon-selective LB medium supplemented with 5 μM beta-alanine at 43° C.,and single colonies tested for retention of chloramphenicol resistance,loss of ampicillin resistance (indicating curing of pKD46), andrequirement for beta-alanine for growth on M9-glucose minimal medium.Confirmation of correct insertion of the CAT gene into the panD locuswas carried out by colony PCR of the resultant ΔpanD::CAT strain usingprimers that flank the insertion locus (TTACCGAGCAGCGTTCAGAG, SEQ ID NO:6; and CACCTGGCGGTGACAACCAT, SEQ ID NO: 7). While the wild-type panDlocus is expected to yield a PCR product of 713 basepairs, theΔpanD::CAT construct yielded a 1215-basepair product. A derivative ofthe ΔpanD::CAT strain, in which the inserted CAT gene is removed by theactivity of the FLP recombinase encoded by plasmid pCP20, wasconstructed as described previously (Datsenko and Wanner, Proc. Natl.Acad. Sci. USA 97: 6640-5, 2000). This strain is referred to as ΔpanD.

A secondary route to beta-alanine exists in E. coli based on thereductive pathway of uracil catabolism (West, Can. J. Microbiol. 44:1106-9, 1998, FIG. 2). In this pathway, uracil is reduced todihydrouracil by the enzyme dihydropyrimidine dehydrogenase (E.C.1.3.1.2). Dihydrouracil is then converted by dihydropyrimidinase (E.C.3.5.2.2) to N-carbamoyl-beta-alanine, which in turn is hydrolyzed byN-carbamoyl-beta-alanine amidohydrolase (E.C. 3.5.1.6) to beta-alanine,CO₂, and NH₃. To prevent the formation of beta-alanine by this pathway,the gene encoding dihydropyrimidine dehydrogenase, yeiA (GenBankAccession No. AAC75208), was insertionally deleted by the method ofDatsenko and Wanner as described above. The CAT gene of pKD3 wasamplified using primersGCGGCGTGAAGTTTCCCAACCCGTTCTGCCTCTCTTCTTCGTGTAGGCTGGAGCTG CTTC (SEQ IDNO: 8), and TTACAACGTTACCGGGTGTTCTTTCTCGCCTTTCTTAAACCATATGAATATCCTCCTTAG (SEQ ID NO: 9), where the underlined sequence corresponds to theregions in the E. coli chromosome immediately upstream and downstream ofthe yeiA locus, respectively, and the non-underlined sequence arehomologous to the regions in pKD3 that permit amplification of afragment containing the CAT gene. Chloramphenicol-resistant insertionmutants were isolated as described above, and the resistance markertransduced into the ΔpanD strain to generate the double mutantΔpanD/ΔyeiA::CAT.

Electrocompetent cells of E. coli BW 25115 ΔpanD::CAT, ΔpanD, orΔpanD/ΔyeiA::CAT, were generated and used as hosts for thetransformation of libraries of mutant lysine 2,3-aminomutase DNAs asdescribed in EXAMPLE 6.

Example 5 Cloning and In Vitro Mutagenesis of a Porphyromonas gingivalisKAM Gene

The lysine 2,3-aminomutase gene from Porphyromonas gingivalis wasamplified by PCR from genomic DNA and cloned into the NdeI and NotIsites of vector pET22B (Novagen). Mutagenic PCR was conducted by themethod of Cadwell and Joyce (PCR Methods Appl. 2:28-33, 1992), using T7promoter and T7 terminator primers for amplification and 6.25 μL or 9.38μL of mutagenic buffer per 100 μL reaction. The PCR products were gelpurified (Qiagen) and digested sequentially with NdeI and NotI. Thedigested PCR products were ligated into pPRONde vector and transformedinto E. coli Electromax™ DH10B™ as described in EXAMPLE 2. Multipletransformations were conducted to obtain at least 60,000 colonies permutation treatment. Colonies were scrapped from plates and plasmid DNAwas prepared and precipitated to increase its concentration. Theresulting libraries had mutation rates of 0.3% and 0.35%.

Example 6 Identification of Clones Having Alanine 2,3-AminomutaseActivity

The mutagenized lysine 2,3-aminomutase plasmid libraries generated abovein EXAMPLE 2 was transformed into electrocompetent E. coli strain DV1cells. Transformants were plated on LB containing 25 μg/ml kanamycin atthe appropriate dilution to obtain an estimate of total transformantsand on M9 minimal medium supplemented with 0.4% glucose, 0.2% VitaminAssay Casamino Acids (DIFCO/Becton Dickinson, Sparks, Md.), and 25 μg/mlkanamycin (Sigma, St. Louis, Mo.). For some selections, IPTG was addedto 0.25 mM.

The ΔpanD::CAT strain of E. coli described in EXAMPLE 4 was transformedin a similar manner with libraries generated in EXAMPLES 2, 3, and 5,except that transformants were plated on M9 minimal medium supplementedwith 0.4% glucose, and 25 μg/ml kanamycin (Sigma, St. Louis, Mo.). Forsome selections on B. subtilis libraries, IPTG was added to 0.25 mM,Fe₂(NH₄)₂SO₄ was added to 50 μM, and chloroamphenicol was added to 25μg/mL. For some selections on P. gingivalis libraries, IPTG was added to50 μM, Fe₂(H₄)₂SO₄ was added to 50 μM, chloroamphenicol was added to 25μg/mL, L-alanine was added to 1 mg/mL and L-lysine was added to 2 mg/mL.

Transformants growing on the minimal medium plates arose at a frequencyof approximately 1×10⁻⁴ relative to the number to total transformants asmeasured by the number of colonies growing on LB plus 25 μg/mlkanamycin. Plasmid DNA from the colonies growing on minimal medium wasprepared using the Qiagen Miniprep kit and retransformed into theΔpanD::CAT strain of E. coli to confirm that the ability to grow in theabsence of added beta-alanine was conferred by a function carried by theplasmid. Plasmid DNA was prepared from retransformed colonies and thekam gene sequenced to determine any changes relative to the wildtype B.subtilis or P. gingivalis kam gene sequences.

A mutated B. subtilis kam gene sequence, which encodes for an alanine2,3-aminomutase, is shown in SEQ ID NO: 20, and the corresponding aminoacid sequence shown in SEQ ID NO: 21. The plasmid carrying this sequenceis designated pLC4-7LC1. There were three amino acid changes observed inthe mutated sequence, as compared to the wildtype B. subtilis kam genesequence (FIG. 4). There was a L103M substitution, a M136V substitution,and a D339H substitution in the alanine 2,3-aminomutase protein (wherethe first amino acid is the wild-type sequence, the number is the aminoacid position, and the second amino acid is the sequence observed in thealanine 2,3-aminomutase sequence). The FeS cluster-binding motif (aminoacids 134-146 of SEQ ID NO: 21) and the putative PLP-binding motif(amino acids of 288-293 SEQ ID NO: 21) are also shown in FIG. 4. This isthe first demonstration of alanine 2,3-aminomutase nucleic acid andamino acid sequences.

A mutated P. gingivalis kam gene sequence, which encodes for an alanine2,3-aminomutase, is shown in SEQ ID NO. 29, and the corresponding aminoacid sequence shown in SEQ ID NO 30. There were five amino acid changesobserved in the mutated sequence, as compared to the wildtype P.gingivalis sequence (FIG. 5). There was a N19Y substitution, a L53Psubstitution, a H85Q substitution, a D331G substitution, and a M342Tsubstitution in the alanine 2,3-aminomutase protein. However, it ispossible that not all of these mutations are necessary to have alanine2,3-aminomutase activity. In aligning the B. subtilis and P. gingivalismutant proteins, the P. gingivalis D331G substitution is located at thecorresponding location within the protein as the B. subtilis D339Hsubstitution (FIG. 6), indicating that it may be of particularimportance. The FeS cluster-binding motif (amino acids 126-138 of SEQ IDNO: 30) and the putative PLP-binding motif (amino acids 280-285 of SEQID NO: 30) are also shown in FIG. 5. This is another demonstration ofalanine 2,3-aminomutase nucleic acid and amino acid sequences.

The ability of the mutated kam genes to convert alpha-alanine tobeta-alanine, and thus to allow production of pantothenate, wasdetermined using liquid growth tests that compare the growth of ΔpanDtransformants in minimal media containing panthothenate with growth inmedia lacking pantothenate. The starting inoculum included washed cellsfrom a growing culture, or cells scrapped from a plate.

As an example of using washed cells as inoculum, 3-5 mL cultures werestarted from single colonies and grown overnight at 30° C. in LB brothplus 40 μg/ml kanamycin. The OD₆₀₀ of the cultures were read andequivalent numbers of cells from each culture harvested bycentrifugation (approximately 600 total OD×μL, e.g. OD 4.0×150 μL). Thecells were washed twice with 0.85% NaCl, resuspended in 200 μL 0.85%NaCl, and 30 μL was used to inoculate 3 ml of M9-based minimal media (6g/L Na₂HPO₄, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 1 g/L NH₄Cl, 2 mM MgSO₄, 4 g/Lglucose, 1 mM CaCl₂, 250 μM IPTG, 40 μg/mL kanamycin, 50 μMFe(NH₄)₂(SO₄)₂) in glass 13 mm diameter tubes to a starting OD₆₀₀ ofabout 0.05. Control cultures contained either minimal media supplementedwith 20 μM pantothenate, or LB broth with 40 μg/mL kanamycin. Cultureswere grown at 37° C. without shaking for about 18 hours and the OD₆₀₀measured.

As shown in Table 1, whereas the cells carrying the vector control grewin minimal medium without added pantothenate or beta-alanine to anaverage OD₆₀₀ of 0.21 using residual reserves of pantothenate orbeta-alanine (approximately 30% of the OD₆₀₀ achieved in the presence ofpantothenate), the cells carrying the alanine 2,3-aminomutase clone grewto an average OD₆₀₀ of 0.50, approximately 90% of that achieved in thepresence of pantothenate. The addition of Fe²⁺ did not increase thegrowth of the vector control cells, but allowed the alanine2,3-aminomutase-bearing cells to achieve growth densities equal to thatobtained in the presence of pantothenate or in rich LB broth medium.This indicates that the alanine 2,3-aminomutase gene provides a sourceof beta-alanine that complements the panD mutation.

TABLE 1 Growth test Medium Minimal + Minimal + Minimal Fe²⁺ pantothenateLB Plasmid Replicate (OD₆₀₀) pPRONde 1 0.206 0.210 0.670 0.647 2 0.2170.209 0.683 0.640 pLC4-7LC1 1 0.458 0.550 0.522 0.572 2 0.460 0.5410.576 0.552

As an example of using plated cultures as inoculum, a colony wasscrapped off a plate and resuspended in 50 μL of minimal media lackingpantothenate. The resuspension (20 μL) was used to inoculate 1 mL ofminimal media in a 1.5 mL microtube and 20 μL was used to inoculate onemL of minimal media supplemented with pantothenate. The latter cultureserved as a control for the varying amount of inoculum added. Theresponse of the culture to growth without pantothenate was expressed asa ratio of growth on media lacking pantothenate to growth on mediacontaining pantothenate. Cultures were grown at 25-37° C. for 1-3 dayswith no shaking and OD₆₀₀ measured. A more anaerobic growth test wasobtained by filling tubes to a greater extent. This was helpful intesting the P. gingivalis mutants.

TABLE 2 Test with scrapped colonies/semi-anaerobic. OD₆₀₀:M9 + RatioOD_(600:)M9/OD₆₀₀:M9 + OD₆₀₀:M9 pantothenate pantothenate Pg aam 0.7110.791 0.90 Pg aam 0.617 0.77 0.80 Pg aam 0.702 0.811 0.87 Pg aam 0.7120.879 0.81 Pg aam 0.689 0.843 0.82 Pg aam 0.719 0.851 0.84 pPRONde 0.1480.824 0.18 Bs aam 0.783 0.801 0.98 Bs aam 0.777 0.838 0.93 Pg kam 0.0640.792 0.08 Pg kam 0.195 0.876 0.22 Pg aam = Cells with plasmid carryingmutated P. gingivalis kam gene with alanine 2,3-aminomutase activity Bsaam = Cells with plasmid carrying mutated B. subtilis kam gene withalanine 2,3-aminomutase activity Pg kam = Cells with plasmid carryingwildtype P. gingivalis kam gene

Example 7 Generation of Individual Mutations in B. subtilis Lysine2,3-Aminomutase

The mutations identified in the wildtype B. subtilis lysine2,3-aminomutase gene above in EXAMPLE 5 were individually constructed inthe wildtype B. subtilis lysine 2,3-aminomutase gene (SEQ ID NO: 3)using the Stratagene QuikChange™ Site-Directed Mutagenesis Kit. Theoligonucleotides used to generate the L103M mutation were:CACAAAACAAAATACGATATGGAAGACCCGCTCCATGAGGATGAAGATTCA (SEQ ID NO: 10), andTGAATCTTCATCCTCATGGAGCGGGTCTTCCATATCGTATTTTGTTTTGTG (SEQ ID NO: 11). Theoligonucleotides used to generate the M136V mutation were:GAATCAATGTTCCGTATACTGCCGCTAC (SEQ ID NO: 12), andGTAGCGGCAGTATACGGAACATTGATTC (SEQ ID NO: 13). The oligonucleotides togenerate the D339H mutation were: GTTCCTACCTTTGTTGTACACGCACCAGGCG (SEQID NO: 14), and CGCCTGGTGCGTGTACAACAAAGGTAGGAAC (SEQ ID NO: 15).

Using the liquid growth test described in EXAMPLE 6, cells with plasmidscarrying the L103M mutation alone were capable of growth in minimalmedium without added pantothenate or beta-alanine, however not to thesame extent as cells with plasmid pLC4-7LC1, whereas those carrying theM136V or D339H mutations alone had the host ΔpanD phenotype.Combinations of the L103M mutation with the M136V and D339H mutations inan otherwise wildtype B. subtilis kam sequence yielded a gene thatconferred the same ability to grow in the absence of beta-alanine orpantothenate as did pLC4-7LC1, confirming that these three mutations, ora subset of them, are sufficient to confer alanine 2,3-aminomutaseactivity.

One skilled in the art will understand that alternative substitutions inthese positions can be generated. Thus, using oligonucleotides similarto SEQ ID NOS: 10 and 11 in which the codon corresponding to L103 wasrandomized, mutants with substitutions L103K, L103R, L103E, and L103Swere obtained that conferred to the ΔpanD strain the ability to grow inthe absence of beta-alanine or pantothenate. Further, usingoligonucleotides similar to SEQ ID NO: 14 and 15 in which the codoncorresponding to D339 was randomized, mutants with substitutions D339Q,D339T, D339N, were obtained that conferred to the ΔpanD strain theability to grow in the absence of beta-alanine or pantothenate.

Example 8 Selection for Alanine 2,3-Aminomutase Activity Without UsingMutagenized Lysine 2,3-Aminomutase

An alternative method to identifying cells having alanine2,3-aminomutase activity is to plate cells, such as the DV1 orΔpanD::CAT cells described above, on the media described above, withouttransfecting them with the mutagenized lysine 2,3-aminomutase library.Such cells are selected as described above, and verified for thepresence of alanine 2,3-aminomutase activity as described in EXAMPLES 6and 9.

Cells can be mutagenized before plating, for example by exposing thecells to UV irradiation or chemicals (such as MES). This permitsisolation of mutants having mutations in one or more other genes whichresult in the cell having alanine 2,3-aminomutase activity.

Alternatively, the cells can be unaltered before plating (e.g. nottransformed, not mutagenized). This method permits isolation ofnaturally occurring strains having alanine 2,3-aminomutase activity.

Example 9 Demonstration of Alanine 2,3-Aminomutase Activity

Cells obtained using the screening methods described above were verifiedfor their alanine 2,3-aminomutase activity. For cells that weretransformed with a mutagenized library (EXAMPLES 2, 3 and 5), plasmidswere isolated from selection host using standard molecular biologymethods. The resulting plasmids were retransformed into the selectionhost, the plasmids reisolated, and the resulting clones sequenced asdescribed in EXAMPLE 6. For un-transformed cells (EXAMPLE 8), the geneconferring the alanine 2,3-aminomutase activity can be cloned, forexample using shotgun cloning.

Several assays can be used to assay for alanine 2,3-aminomutaseactivity, such as measuring biosynthesis of [¹³C]coenzyme A from[3-¹³C]alanine via [3-¹³C]beta-alanine, by using an enzyme assay thatmeasures the conversion of alpha-alanine to beta-alanine, or an assaythat measures the presence of beta-alanine in cells or extracts of cellscarrying alanine 2,3-aminomutase.

Biosynthesis of [¹³C]Coenzyme A from [3-¹³C]Alpha-Alanine Via[3-¹³C]Beta-Alanine

Insertional deletion of the panD gene, whose gene product (aspartate1-decarboxylase) catalyzes the production of beta-alanine fromaspartate, results in pantothenate deficiency and hence the inability toproduce coenzyme A. However, ΔpanD cells possessing an alanine2,3-aminomutase capable of producing beta-alanine from alpha-alaninewould be able to bypass this deficiency; in particular, these cells,when grown in the presence of [3-¹³C]alpha-alanine, would incorporatethe [¹³C] label into coenzyme A. This test was used to confirm that theB. subtilis alanine 2,3-aminomutase sequence isolated in EXAMPLE 6 (SEQID NOS: 20 and 21) could catalyze the conversion of alpha-alanine tobeta-alanine.

Cells of E. coli ΔpanD/AyeiA::CAT transformed with pPRONde, pPRO-KAM1,or pLC4-7LC1 were grown overnight at 37° C. in minimal medium (EXAMPLE6) except with 25 μg/ml kanamycin and 10 μM Fe(NH₄)₂(SO₄)₂, and with 1mM alanine (unlabeled), and 10 μM beta-alanine. The cultures werediluted 100-fold in minimal medium with 25 μg/ml kanamycin, 10 μMFe(NH₄)₂(SO₄)₂, and 11 mM [3-¹³C]alpha-alanine (99%, Cambridge IsotopeLaboratories, Andover, Mass.) but no unlabeled alpha-alanine orbeta-alanine. Following growth at 30° C. for approximately 20 hours, thecells were recovered by centrifugation and extracts generated by themethod of Jaskowski and Rock (J. Bacteriol. 148: 926-32, 1981) in thepresence of 10 mM dithiothreitol to convert thioesters of coenzyme A tothe free sulfhydryl form.

The extracts were analyzed using a Micromass Ultima LC/MS system whichincluded of a Waters 2690 liquid chromatograph with a Waters 996Photo-Diode Array (PDA) absorbance monitor placed in series between thechromatograph and the triple quadrupole mass spectrometer. LCseparations were made using a 4.6×150 mm YMC ODS-AQ (3 μm particles, 120Å pores) reversed-phase chromatography column at room temperature.Gradient elution of the analytes was performed using aqueous 25 mMammonium acetate containing 0.5% (v/v) acetic acid (Buffer A), andacetonitrile containing 0.5% (v/v) acetic acid (Buffer B). The elutionwas isocratic at 10% B, 0-10 min, then linear from 10% B to 100% B,10-12 min. The flow rate was 0.250 mL/min and photodiode array UVabsorbance was monitored from 200 nm to 400 nm. All parameters of theelectrospray MS system were optimized and selected based on generationof protonated molecular ions ([M+H]⁺) of the analytes of interest, andproduction of characteristic fragment ions. The following instrumentalparameters were used for ESI-MS detection of Coenzyme A in the positiveion mode: capillary: 4.0 V; cone: 80 V; hex 1: 25 V; aperture: 0 V; hex2: 0 V; source temperature: 100° C.; desolvation temperature: 350° C.;desolvation gas: 500 L/h; cone gas: 40 L/h; low mass resolution: 15.0;high mass resolution: 15.0; ion energy: 0; multiplier: 650.Uncertainties for reported mass/charge ratios (m/z) and molecular massesare ±0.01%. The ratio of peak areas with m/z 769 ([¹³C]coenzyme A) topeak area with m/z 768 (unlabeled coenzyme A) are shown in Table 3.

TABLE 3 Biosynthesis of [¹³C]Coenzyme A Plasmid or sample Ratio m/z =769:m/z = 768 Coenzyme A standard 0.26 pPRONde 0.36 pPRO-KAM1 0.53pLC4-7LC1 1.90

The results shown in Table 3 confirm that cells bearing the plasmidcarrying the mutant with alanine 2,3-aminomutase activity (SEQ ID NOS:20 and 21), when grown on [¹³C]alpha-alanine, produce a higher ratio of[¹³C]coenzyme A to [¹²C]coenzyme A compared to normal abundance[¹³C]coenzyme A or with cells bearing either the vector or the wildtypeB. subtilis lysine 2,3-aminomutase gene. This demonstrates that thealanine 2,3-aminomutase sequence can produce beta-alanine, an obligatoryintermediate in the biosynthesis of coenzyme A.

Enzyme Assays

An enzyme assay which measures the conversion of alpha-alanine tobeta-alanine, or which measures for the presence of beta-alanine, can beperformed to determine if a cell has alanine 2,3-aminomutase activity.For example, the method described by Chen et al. (Biochem. J.348:539-49, 2000) to determine the lysine 2,3-aminomutase activity canbe applied to the determination of alanine 2,3-aminomutase activity bysubstituting L-[U-¹⁴C]alanine for L-[U-¹⁴C]lysine in the incubation withreductively preincubated enzyme or cell extract, and separation of theradioactive alpha-alanine and beta-alanine by paper electrophoresisfollowed by scintillation counting of the spots corresponding toalpha-alanine and beta-alanine, respectively. Alternatively, thepurified and reductively preincubated alanine 2,3-aminomutase can beincubated with alpha-alanine and the reaction mixture separated by highperformance liquid chromatography to separate the product beta-alaninefrom alpha-alanine and quantify the product (Abe et al., J.Chromatography B, 712:43-9, 1998).

The formation of beta-alanine from alpha-alanine can also be monitoredin whole cells of the E. coli ΔpanD::CAT strain transformed with aplasmid expressing an alanine 2,3-aminomutase by incubation of the cellsin M9 minimal medium (Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y., 1989) containing 0.4% (w/v) glucose,25 μg/ml kanamycin (for a plasmid conferring resistance to kanamycin),0.25 mM IPTG, and 1 mg/ml [¹³C]-labeled alpha-alanine, extracting thecells, and detecting the [¹³C]-beta alanine by high performance liquidchromatography/mass spectrometry using methods known by those skilled inthe art.

Example 10 Synthetic Operons for 3-HP Production from Beta-Alanine

Biosynthetic pathways that allow production of 3-HP via beta-alaninewere generated (FIG. 1). One pathway to 3-HP from beta-alanine involvesthe use of a polypeptide having CoA transferase activity, that is, anenzyme from a class of enzymes that transfers a CoA group from onemetabolite to the other. As shown in FIG. 1, beta-alanine can beconverted to beta-alanyl-CoA using a polypeptide having CoA transferaseactivity and CoA donors such as acetyl-CoA or propionyl-CoA.Alternatively, beta-alanyl-CoA can be generated by the action of apolypeptide having CoA synthetase activity. The beta-alanyl-CoA can bedeaminated to form acrylyl-CoA by a polypeptide having beta-alanyl-CoAammonia lyase activity. The hydration of acrylyl-CoA at the betaposition to yield 3-HP—CoA can be carried out by a polypeptide having3-HP—CoA dehydratase activity. The 3-HP—CoA can act as a CoA donor forbeta-alanine, a reaction that can be catalyzed a polypeptide having CoAtransferase activity, thus yielding 3-HP as a product. Alternatively,3-HP—CoA can be hydrolyzed to yield 3-HP by a polypeptide havingspecific CoA hydrolase activity.

These pathways use several enzymes that were cloned and expressed asdescribed in WO 02/42418 (herein incorporated by reference) or below.Megasphaera elsdenii cells (ATCC 17753), Chloroflexus aurantiacus cells(ATCC 29365), Clostridium propionicum (ATCC 25522), Clostridiumacetobutylicum (ATCC 824), Pseudomonas aeruginosa (ATCC 17933), Bacillussubtilis (ATCC 23857), Alcaligenes faecalis (ATCC 25094), and rat cDNA(Clontech, Palo Alto, Calif.) were used as sources of DNA. One skilledin the art will understand that similar methods can be used to obtainthe sequence of these enzymes from any organism.

Individual genes were cloned, expressed and assayed prior to operonconstructions. The synthetic operons for production of 3-HP in E. coliwere cloned into pET-11a (5.7 kb) expression vector under control of theT7 promoter (Novagen), pPROLar.A (2.6 kb) vector with lac/ara-1 promoter(Clontech, Palo Alto, Calif.), and pTrc99A (4.2 kb) vector with trcpromoter (Pharmacia Biotech, Uppsala, Sweden). Several operons withdifferent combinations of relevant genes were generated as describedbelow. Assays for propionyl-CoA transferase and acrylyl-CoA hydratase(or 3-HP dehydratase) are described in WO 02/42418 (herein incorporatedby reference).

Isolation of a 3-HP Dehydrogenase Gene from Alcaligenes faecalis M3A

A. faecalis (ATCC #700596) is salt marsh bacterium that metabolizesacrylate via 3-HP. The 3-HP produced from acrylate is likely convertedto malonic semialdehyde by a 3-HP dehydrogenase. To isolate the geneencoding this dehydrogenase, A. faecalis genomic DNA was isolated asfollows. A five-mL A. faecalis culture was grown at 37° C. in trypticasesoy broth, and cells harvested then resuspend in 400 mL TE buffer.Subsequently, 20 mL of 10% SDS, 100 μl 10 mg/ml proteinase K, and 10 mL100 mg/ml lysozyme were added to the cell suspension and the mixtureincubated for two hours at 42° C. with occasional mixing. To this mix,150 mL phenol was added and the mixture shaken for at least two hours at37° C., then approximately 800 mL chloroform added. The mixture wasmixed by vortexing and centrifuged for 30 minutes at 15000 rpm. Theupper aqueous phase was transferred to a clean microfuge tube, and theDNA precipitated with 60 mL 3M NaOAc and approximately 1 mL ethanol, andrecovered by spooling. The DNA was resuspended with 400 mL TE buffer and20 mL 200 mg/ml RNase added, and the mixture incubated for 1 hour at 37°C. The DNA was re-precipitated with NaOAC and ethanol, rinsed severaltimes with 70% ethanol and resuspend in TE buffer.

The following degenerate primers were designed based on conservedregions of publicly known amino acid sequences of the3-hydroxyisobutrate dehydrogenase genes which are expected to behomologous to the desired 3-HP dehydrogenase. AFHPDHF1: 5′TTYATYGGBYTSGGBAAYATGGG 3′ (SEQ ID NO: 16); AFHPDHF2: 5′GAYGCNCCNGTBWSSGGBGG 3′ (SEQ ID NO: 17); and AFHPDHR2: 5′CATRTTRTTRCARATYTTNGC 3′ (SEQ ID NO: 18). PCR reactions using A.faecalis genomic DNA as template were carried out using Taq DNApolymerase (Roche) according to the manufacturer's instructions, usingeither SEQ ID NOS: 16 and 18 (reaction A) or SEQ ID NOS: 17 and 18(reaction B). The PCR program consisted of an initial incubation of 94°C. for 2 minutes, 4 cycles of 94° C., 30 seconds; 56° C., 45 seconds,72° C. 3 minutes; 4 cycles of 94° C., 30 seconds; 54° C., 45 seconds,72° C. 3 minutes; 4 cycles of 94° C., 30 seconds; 52° C., 45 seconds,72° C. 3 minutes; 4 cycles of 94° C., 30 seconds; 50° C., 45 seconds,72° C. 3 minutes; and 16 cycles of 94° C., 30 seconds; 47° C., 45seconds, 72° C. 3 minutes, followed by a final incubation of 7 minutesat 72° C. Both reactions gave products of approximately 500 bp. The PCRproduct from reaction A was gel isolated, cloned into pCR11 andtransformed into TOP10 chemical competent cells, selected on LB mediumcontaining 50 mg/ml kanamycin. Clones having the right size insert wereselected and their plasmids isolated and sequenced. Based on thesesequences the following gene specific nested primers were designed:GWHPDF1: 5′ GGTTTACGAGGGCGAGAACGGCTTGCT 3′ (SEQ ID NO: 19); GWHPDF2: 5′CAAGCTGGGTCTGTTCATGCTGGATG 3′ (SEQ ID NO: 26); GWHPDR1: 5′AAGCGGTTCTCGCCCTCGTAAACCTGA 3′ (SEQ ID NO: 27); and GWHPDR2: 5′CGCATTCAAGTCAAAGACGTTCAGGCTA 3′ (SEQ ID NO: 32) and the Genome Walktechnique used to isolate the entire ORF of the gene encoding the 3-HPdehydrogenase. The sequence of this ORF is shown in SEQ ID NO: 33, andthe corresponding protein sequence in SEQ ID NO: 34. The start codon ofthe 3-HP dehydrogenase is at position 408 of SEQ ID NO: 33 and ispreceeded by a ribosome-binding site at positions 397-403 of SEQ ID NO:33. The stop codon is at position 1304 of SEQ ID NO: 33.

Cloning, Expression, and Assay of β-Alanine-CoA Ammonia Lyase (ACL)

Two acl genes were cloned from C. propionicum. acl-1 (SEQ ID NO: 22)encodes a 145 amino acid protein (SEQ ID NO: 23) and acl-2 (SEQ ID NO:53) encodes a 144 amino acid protein (SEQ ID NO: 54). These two proteinsare highly homologous and differ by only 8 amino acids at theC-terminus. The acl-1 and acl-2 genes were cloned using the followingprimers: OSaclNdeF: 5′-GGGAATTCCATATGGTAGGTAAAAAGGTTGTACATC-3′ (SEQ IDNO: 35), and OSaclBamR: 5′-CGACGGATCCATTCGTCCGCTTGAATAACTAAAG-3′ (SEQ IDNO: 36) for acl-1, and SEQ ID NO: 35 and OSac12BamR:5′-CGACGGATCCCGAAAATGTCACCAAAAATTATTGAG-3′ (SEQ ID NO: 37) for acl-2.The resulting sequences were cloned into the pET11a vector digested withNdeI and BamHI. Resulting plasmids pACL-1 and pACL-2 were transformedinto BL21(DE3) cells. BL21(DE3) carrying pET11a (control), pACL-1 andpACL-2 were grown in 10 ml LB medium supplemented with 50 μg/mlcarbenicillin to OD₆₀₀˜0.5 and induced with 100 μM IPTG for 4 hours. Theinduced cells were collected by centrifugation at 3500 rpm in Avanti J20centrifuge (Beckman, Fullerton, Calif.) and treated with Bug Buster(Novagen, Madison, Wis.) according to the manufacturer instruction. Theresulting cell extract was used in an enzyme assay that followed theconversion of acrylyl-CoA to beta-alanine-CoA (the reverse reaction withrespect to the pathway in FIG. 1).

The assay mixture contained 10 μl 1M TAE, 20 μl 1M NH₄Cl, 2 μl 100 μMacrylyl-CoA, 10 μl cell extract, and 158 μl H₂O. The enzymatic reactionwas incubated for five minutes at 37° C. and stopped by addition of 200μl 10% TFA. The mixture was loaded on C18 Sep-Pak Vac Icc column(Waters, Milford, Mass.), eluted with 200 μl 40% acetonitrile, 0.1% TFAand reduced in volume to 100 μl by centrifugation in SpeedVac (SavantInstruments, Holbrook, N.Y.). Formation of beta-alanyl-CoA was detectedby LC-MS using standard methods. Both ACL-1 and ACL-2 enzymes wereactive and used for beta-alanine operon construction.

Cloning, Expression, and Assay of CoS Transferase from E. coli

The open reading frame yfdE (identified as a hypothetical protein in thePubMed database) was amplified using PCR. Because the open reading framehad two potential start sites, the following primers were used to cloneand express both genes: yfdE gtg nde sen(5′-AGAGAGCATATGTCTTTTCACCTTCGGC-3′; SEQ ID NO: 38), and yfdE atg ndesen (5′-AGAGAGGGATCCGCGGCTCCCACAATGTTGAAATG-3′ SEQ ID NO: 39) foryfdE-1, and yfdE gtg nde sen (SEQ ID NO: 38) and yfdE bam anti(5′-AGAGAGCATATGACAAATAATGAAAGCAAAGG-3′, SEQ ID NO: 40) for yfdE-2.

Chromosomal DNA from E. coli MG1655 was used as template for PCRperformed with Pfu Turbo (Stratagene) using the following PCRconditions: 94° C. for 5 minutes; 25 cycles of 94° C. for 30 seconds,55° C. for 30 seconds, and 72° C. for 2 minutes 20 seconds, followed byincubation at 72° C. for 7 minutes. The PCR reaction was purified usingQIAquick PCR purification Kit (Qiagen), digested with NdeI and BamHI,cloned into pET28b (Novagen) digested with the same restriction enzymes,and transformed into chemically competent TOP10 cells (Invitrogen).Plasmids from positive clones were isolated and transformed intoBL21(DE3) expression cells (Novagen). Cells were grown in LB media at37° C. to an OD₆₀₀ of 0.6 and were induced with 100 μM IPTG andincubated an additional 3 hours after induction. The cells wereharvested by centrifugation and washed once with 0.85% NaCl. The cellpellet was stored at −80° C. until further use.

The cell pellet was thawed on ice, and resuspended in 4 ml bindingbuffer (Novagen HisBind purification kit). The cells were lysed by threepassages through a French Pressure Cell (SLM Aminco) (10000 psi). Thecell debris was removed by centrifugation (30,000×g for 30 minutes). Theextract was filtered through a 0.45 μm syringe filter before loading ona Quick 900 cartridge following the manufacturer's instructions(Novagen). Purified protein was desalted using a PD-10 column(Pharmacia) according to the manufacturer's instructions. The bufferused was 5 mM boric acid, 5 mM Tris, 5 mM citric acid, 5 mM NaH₂PO₄ pH7.0.

Purified protein was assayed using a reaction mix containing 100 mM Kphosphate, pH 7.0, 100 mM beta-alanine, 1 mM acetyl-CoA, and 20 μlpurified CoA transferase in a total assay volume of 200 μl. The reactionwas incubated for 20 minutes at room temperature and then stopped with100 μl 10% trifluoroacetic acid (TFA). The reactions were purified using1 cc SepPak Vac cartridges (Waters Milford, Mass.) conditioned with 1 mlmethanol and washed twice with 1 ml 0.1% TFA. The sample was applied andthe cartridge washed twice with 1 ml 0.1% TFA. The sample was elutedwith 200 μl 40% acetonitrile containing 0.1% TFA, dried to ½ volume in arotary evaporator and analyzed by liquid chromatography/massspectrometry. A peak corresponding to the expected mass forbeta-alanyl-CoA was present in assays with yfdE-1 or yfdE-2 proteins,and this peak was not present in the controls omitting the purifiedproteins, indicating that the CoA transferases are responsible for thesynthesis of beta-alanyl-CoA.

Operons 1 and 2: ACL—Propionyl-CoA Transferase—Acrylyl-CoA Hydratase

Operons for the following conversion: beta-alanine to beta-alanyl-CoA toacrylyl-CoA to 3-HP were constructed. A gene encoding CoA transferasewas amplified from genomic DNA of M. elsdenii by PCR with OSNBpctF(5′-GGGAATTCCATATGAGAAAAGTAGAAATCATTACAGCTG-3′; SEQ ID NO: 41) and OSHTR(OSHTR: 5′-ACGTTGATCTCCTTCTACATTATTTTTTCAGTCCCATG-3′; SEQ ID NO: 42)primers.

A CoA hydratase gene was amplified from genomic DNA of C. aurantiacus byPCR with OSTHF (5′-CATGGGACTGAAAAAATAATGTAGAAGGAGATCAACGT-3′; SEQ ID NO:43) and OSHBR (5′-CGACGGATCCTCAACGACCACTGAAGTTGG-3′; SEQ ID NO: 44)primers.

ACL-1 and ACL-2 (beta-alanine-CoA ammonia lyase) genes were amplifiedfrom C. propionicum genomic DNA with primer pairs OsaclXbaF(5′-CTAGTCTAGAGCTTTCTAAGAAACGATTTCCG-3′; SEQ ID NO: 45) and OSaclNdeR(5′-GGGAATTCCATATGCGTAACTTCCTCCTGCTATCATTCACCGGGGTGCTTTCT-3′; SEQ ID NO:46) for acl-1; and OSac12XbaF (5′-CTAGTCTAGAGGAAACCGCTTAACGAACTC-3′; SEQID NO: 47) and OSac12-2NdeR(5′-GGGAATTCCATATGCGTAACTTCCTCCTGCTATTATTGAGGGTGCTTTGCATCC-3′; SEQ IDNO: 48) for acl-2.

PCR was conducted in a Perkin Elmer 2400 Thermocycler using Pfu Turbopolymerase (Stratagene) according to the manufacturer instructions. PCRwas performed under the following conditions: initial denaturation step94° C. for 2 minutes; 25 cycles of 94° C. for 30 seconds, 55° C. for 30seconds, 72° C. for 2 minutes; final extention at 72° C. for 7 minutes.Resulting PCR products were gel purified using Qiagen Gel Extraction Kit(Qiagen, Inc.).

CoA-transferase and CoA-hydratase PCR products were assembled togetherin assembly PCR. OSTHF and OSHTR primers (SEQ ID NOS: 42 and 43) arecomplementary to each other which allowed the complementary DNA ends toanneal to each other during PCR and to extend the DNA in bothdirections. To ensure the efficiency of the assembly and the followingamplification, two end primers OSNBpctF and OSHBR (SEQ ID NOS: 41 and44) were added to the assembly PCR mixture containing 100 ng of thepurified CoA-transferase and CoA-hydratase PCR products and the mix ofrTth polymerase (Applied Biosystems, Foster City, Calif.) and Pfu Turbopolymerase (Stratagene) in a ratio of 8:1. The polymerase mix ensuredhigher fidelity of the PCR reaction. Assembly PCR was run under thefollowing conditions: initial denaturation step 94° C. for 1 minute; 20cycles of 94° C. for 30 seconds, 54° C. for 30 seconds, 68° C. for 2.5minutes; final extention at 68° C. for 7 minutes. The assembled PCRproduct was gel purified as described above and digested with NdeI andBamHI. The sites for these restriction enzymes were introduced toassembled PCR product with OSNBpctF (NdeI) and OSHBR (BamHI) primers(SEQ ID NOS: 41 and 44). The digested PCR product was incubated at 80°C. for 30 minutes (to inactivate the restriction enzymes) and useddirectly for ligation to pET11a vector.

Vector pET11a was digested with NdeI and BamHI, gel purified usingQiagen Gel Extraction kit, treated with shrimp alkaline phosphatase assuggested by the manufacturer (Roche Molecular Biochemicals) and usedfor ligation with the assembled PCR product. Ligation was performed at16° C. overnight using T4 ligase (Roche Molecular Biochemicals). Theligation mixture was transformed into chemically competent NovaBluecells (Novagen) and plated on LB plates supplemented with 50 μg/mlcarbenicillin. Individual colonies were selected for plasmid DNApurification; plasmid DNA was obtained using Qiagen Spin Miniprep Kit.Plasmids were digested with NdeI and BamHI and analyzed by gelelectrophoresis.

The resulting plasmid was named pTH, digested with XbaI and NdeI,purified using gel electrophoresis and Qiagen Gel Extraction kit asdescribed above, and used as a vector for consequent cloning of ACL-1and ACL-2 PCR products digested with the same enzymes. The ligation wasperformed as described above, and the ligation mixture transformed intochemically competent NovaBlue cells as described above. Individualcolonies were selected for plasmid DNA purification; plasmid DNA wasobtained using Qiagen Spin Miniprep Kit. Plasmids were digested withXbaI and NdeI and analyzed by gel electrophoresis. Resulting pATHplasmids carrying the constructed operon were transformed into E. coliBL21 (DE3) cells to determine the expression of the cloned genes.

To measure the gene expression and 3-HP production, BL21(DE3) cellscarrying pATH-1 and pATH-2 plasmids were grown to OD₆₀₀˜0.5 in M9CAmedium (Difco Laboratories, Sparks, Mass.) supplemented with 10 g/lglucose, 5 g/l beta-alanine and 50 μg/ml carbenicillin, and induced with100 μM IPTG under aerobic conditions. BL21(DE3) cells carrying pET11avector served as a control. Cell samples were taken 2 and 4 hours afterIPTG induction for polyacrylamide gel electrophoresis analysis. Allthree enzymes were expressed as shown by the appropriate sized band onthe gel. Production of 3-HP from beta-alanine was detected with bothoperon constructs, pATH-1 and pATH-2, but not in the control cells byLC-MS analysis.

Operon 3: 4-Aminobutyrate Aminotransferase—3-HydroxyisobutyrateDehydrogenase

In an alternative or additional pathway, beta-alanine can be deaminatedby a polypeptide having beta-alanine-2-oxoglutarate aminotransferaseactivity to yield malonate semialdehyde, which can be further reduced to3-HP by a polypeptide having 3-HP dehydrogenase activity or apolypeptide having 3-hydroxyisobutyrate dehydrogenase activity.

Methods for isolating, sequencing, expressing, and testing the activityof such polypeptides are described in WO 02/42418 (herein incorporatedby reference). One skilled in the art will understand that similarmethods can be used to obtain the sequence of any such polypeptide fromany organism.

The gene encoding 4-aminobutyrate aminotransferase was amplified fromgenomic DNA of C. acetobutylicum by PCR with OsabatF(5′-CCGGAATTCTTTAATATGCGATTTGGAGGAG-3′; SEQ ID NO: 49) and OSDATR(5′-GTCCGTCTCCCTTTCAGCTTAAATCGCTATTCTTATAGC-3′; SEQ ID NO: 50) primers.A gene encoding 3-hydroxyisobutyrate dehydrogenase was amplified fromgenomic DNA of P. aeruginosa by PCR with OSATDF(5′-GCTATAAGAATAGCGATTTAAGCTGAAAGGGAGACGGAC-3′; SEQ ID NO: 51) andOSibdR (5′-CGACGGATCCGCAGTGAGTGAGCCTTGGAG-3′; SEQ ID NO: 52) primers.PCR was conducted in a Perkin Elmer 2400 Thermocycler using Pfu Turbopolymerase according to the manufacturer instructions under thefollowing conditions: initial denaturation step 94° C. for 2 minutes; 25cycles of 94° C. for 30 seconds, 56° C. for 30 seconds, 72° C. for 1.5minutes; final extention at 72° C. for 10 minutes. Resulting PCRproducts were gel purified using Qiagen Gel Extraction Kit.

PCR products of 4-aminobutyrate aminotransferase and3-hydroxyisobutyrate dehydrogenase were assembled together in assemblyPCR. The primers shown in SEQ ID NOS: 50 and 51 are complementary toeach other and therefore complementary DNA ends could anneal to eachother during PCR reaction and to extend the DNA in both directions. Toensure the efficiency of the assembly and the following amplification,two end primers OSabatF and OSibdR (SEQ ID NOS: 49 and 52) were added tothe assembly PCR mixture containing 100 ng of the purified4-aminobutyrate aminotransferase and 3-hydroxyisobutyrate dehydrogenasePCR products and the mix of rTth polymerase and Pfu Turbo polymerase in8:1 ratio. Assembly PCR was run under the following conditions: initialdenaturation step 94° C. for 1 minute; 25 cycles of 94° C. for 30seconds, 55° C. for 30 seconds, 68° C. for 3 minutes; final extention at68° C. for 7 minutes. The assembled PCR product was gel purified asdescribed above and digested with EcoRI and BamHI. The sites for theserestriction enzymes were introduced to assembled PCR product withOSabatF (EcoRI) (SEQ ID NO: 49) and OSibdR (BamHI) (SEQ ID NO: 52)primers. The digested PCR product was heated at 80° C. for 30 minutes,gel purified using Qiagen Gel Extraction kit, and used for ligation topPROLar.A vector.

pPROLar.A was digested with EcoRI and BamHI, gel purified using QiagenGel Extraction kit, treated with shrimp alkaline phosphatase assuggested by the manufacturer and used for ligation with the assembledPCR product. The ligation was performed as described above andtransformed into chemically competent TOP10 cells (Novagen) and platedon LB plates supplemented with 25 μg/ml kanamycin. Individual colonieswere selected for plasmid DNA purification; plasmid DNA was obtainedusing Qiagen Spin Miniprep Kit. Plasmids were digested with EcoRI andBamHI and analyzed by gel electrophoresis. The resultant plasmidcarrying the 4-aminobutyrate aminotransferase and 3-hydroxyisobutyratedehydrogenase genes was designated pATD.

To observe gene expression and 3-HP production, TOP 10 cells carryingpATD plasmids or without plasmids (control) were grown to OD₆₀₀˜0.5 inLB medium supplemented with 5 g/l glucose, 5 g/l beta-alanine and 25μg/ml kanamycin and induced with 100 μM IPTG and 0.5% arabinose underaerobic conditions. Production of 3-HP from beta-alanine was detectedwith cells carrying pATD, but not in the control cells by LC-MSanalysis. 3-HP was observed in cell supernatants after 6 and 24 hours ofIPTG induction.

Example 11 Synthetic Operons for 3-HP Production from Alpha-Alanine

Several operons were generated in EXAMPLE 10 which permit production of3-HP via beta-alanine through several alternative pathways. The methodsdisclosed in this example expand on that, by including the disclosedalanine 2,3-aminomutase sequences disclosed herein in an operon. Thisallows production of 3-HP via alpha-alanine.

Operon 4: Alpha-Alanine Aminomutase—Acl—Propionyl-CoATransferase—Acrylyl-CoA Hydratase

An operon for the conversion of alpha-alanine to beta-alanine tobeta-alanyl-CoA to acrylyl-CoA to 3-HP was constructed as follows.Plasmid pLC4-7LC1 plasmid carrying alanine 2,3-aminomutase (EXAMPLE 6;SEQ ID NOS: 20 and 21) was used for the construction of pLCATH2-1. TheATH-2 operon was amplified from pATH-2 (EXAMPLE 10) with the followingprimers: OSac12NotF2: 5′-AAGGAAAAAAGCGGCCGCAGATTAAAGGAGGAATTCTCAATGG-3′(SEQ ID NO: 55) and OShydXbaR: 5′-CTAGTCTAGATCAACGACCACTGAAGTTGG-3′ (SEQID NO: 56). PCR was conducted as described above using the mix of rTthpolymerase and Pfu Turbo polymerase in 8:1 ratio under the followingconditions: initial denaturation step 94° C. for 2 minutes; 25 cycles of94° C. for 30 seconds, 56° C. for 30 seconds, 68° C. for 2 minutes;final extention at 68° C. for 7 minutes.

The resulting PCR product was purified using Qiagen PCR Purification Kitand digested with NotI and XbaI. Digested DNA was heated at 65° C. for30 minutes for enzyme inactivation, gel purified using Qiagen GelExtraction Kit, and cloned into pLC4-7LC-1 plasmid digested with thesame enzymes. The ligation was performed at 16° C. overnight using T4ligase. The ligation mixture was transformed into chemically competentTuner cells (Novagen) and plated on LB plates supplemented with 25 μg/mlkanamycin. Individual colonies were selected for plasmid DNApurification; plasmid DNA was obtained using Qiagen Spin Miniprep Kit.Plasmids were digested with NotI and XbaI and analyzed by gelelectrophoresis. Resulting Tuner (pLCATH2-1) cells were used to observeexpression of the cloned genes and production of 3-HP from alpha-alanineand beta-alanine.

Operon 5: α-Alanine Aminomutase-4-AminobutyrateAminotransferase-3-Hydroxyisobutyrate Dehydrogenase

An operon for the conversion of alpha-alanine to beta-alanine to malonicsemialdehyde to 3-HP was constructed as follows. Plasmid pLC4-7LC1(EXAMPLE 6) carrying alanine 2,3-aminomutase (EXAMPLE 6; SEQ ID NOS: 20and 21) was used for the construction of pLCATD1. The ATD operon wasamplified from pATD plasmid (EXAMPLE 10) with: OSabatNotF:5′-AAGGAAAAAAGCGGCCGCTTTAATATGCGATTTGGAGGAG-3′ (SEQ ID NO: 57) andOsibdXbaR: 5′-CTAGTCTAGAGCAGTGAGTGAGCCTTGGAG-3′ (SEQ ID NO: 58). PCR wasconducted as described above for operon 4, and the resulting PCR productpurified, digested with NotI and XbaI, cloned into pLC4-7LC-1 plasmid,transformed into chemically competent Tuner cells, and individualcolonies selected as described for Operon 4.

Induction of Operons and 3-HP Production

To observe gene expression and 3-HP production, Tuner cells carryingpLCATH2-1, pLCATD1 plasmids or pPROLar vector (control) were grown toOD₆₀₀˜0.5 in LB medium supplemented with 5 g/l glucose, 5 g/lalpha-alanine or 5 g/l beta-alanine and 25 μg/ml kanamycin and inducedwith 100 μM IPTG and 0.5% arabinose under aerobic conditions. Productionof 3-HP from beta-alanine was detected with cells carrying pLCATH2-1 andpLCATD1, but not in the control cells by LC-MS analysis. 3-HP wasobserved in cell supernatants after 22 hours of induction.

Operon 6: Alanine Aminomutase—Beta-Alanine Aminotransferase-3-HPDehydrogenase—Alpha-Alanine Aminotreansferase

An operon for the conversion of pyruvate to alpha-alanine tobeta-alanine to malonic semialdehyde to 3-HP was constructed as follows.The gene encoding for alanine 2,3-aminomutase was amplified frompLC4-7LC1 by PCR with KAM10F (5′-CACACAGAATTCATTAAAGAGGAG-3′; SEQ ID NO:59) and KAMRBATR5′-CATAATCAAACTCAAAGTCAACCATATAAGATCTCCTCCTTACTTCATGAAGAATC CCCTCC-3′;SEQ ID NO: 60) primers. A beta-alanine aminotransferase gene wasamplified from rat cDNA by PCR with KAMRBATF(5′-GGAGGGGATTCTTCATGAAGTAAGGAGGAGATCTTATATGGTTGACTTTGAGTTT GATTATG-3′;SEQ ID NO: 61) and RBATAFDR(5′-CGTGTTACTCATTTTGTCTCCTCGTCATTTACTTGAAGTCTGCTAAGATAC-3′ (SEQ ID NO:62) primers. 3-HP dehydrogenase was amplified from A. faecalis genomicDNA by PCR with RBATAFDF(5′-GTATCTTAGCAGACTTCAAGTAAATGACGAGGAGACAAAATGAGTAACACG-3′; SEQ ID NO:63) and AFDRAATR(5′-TCATTCACCCGTGAGGCCATGAATATATCTCCTTCTTAAGCTTAGTGCTTCTGACG GTAC-3′;SEQ ID NO: 64) primers. Alpha-alanine aminotransferase gene wasamplified from rat cDNA with AFDRAATF(5′-GTACCGTCAGAAGCACTAAGCTTAAGAAGGAGATATATTCATGGCCTCACGGGTG AATGA-3′;SEQ ID NO: 65) and RATGPTOR (5′-GACTAGATATCTCAGGAGTACTCATGGGTGAA-3′ (SEQID NO: 66) primers.

PCR was conducted as described above under the following conditions:initial denaturation step of 94° C. for 2 minutes; 10 cycles of 94° C.for 30 seconds, 48° C. for 30 seconds, 72° C. for 2 minutes; 5 cycles of94° C. for 30 seconds, 52° C. for 30 seconds, 72° C. for 2 minutes; 10cycles of 94° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for 2minutes; final extention at 72° C. for 7 minutes. PCR products were gelpurified using Qiagen Gel Extraction Kit.

PCR products of alanine 2,3-aminomutase and beta-alanineaminotransferase, as well as PCR products of 3-HP dehydrogenase andalpha-alanine aminotransferase, were assembled as pairs in two assemblyPCR. Primer pairs SEQ ID NOS: 60 and 61, as well as SEQ ID NOS: 64 and65 were complementary to each other and therefore complementary DNA endscould anneal to each other during PCR reaction and extend the DNA inboth directions. To ensure the efficiency of the assembly and thefollowing amplification, two end primers (SEQ ID NOS: 59 and 62) wereadded to the assembly PCR mixture containing 100 ng of two purifiedalanine aminomutase and beta-alanine aminotransferase PCR products andthe mix of rTth polymerase and Pfu Turbo polymerase in a ratio of 8:1.Other two end primers, SEQ ID NOS: 63 and 66 were added to the assemblyPCR mixture containing 100 ng of purified 3-HP dehydrogenase andalpha-alanine aminotransferase, and the mix of rTth polymerase and PfuTurbo polymerase in a ratio of 8:1. Assembly PCR was run under thefollowing conditions: initial denaturation step 94° C. for 2 minutes; 5cycles of 94° C. for 30 seconds, 48° C. for 30 seconds, 68° C. for 4minutes; 5 cycles of 94° C. for 30 seconds, 52° C. for 30 seconds, 68°C. for 4 minutes; 5 cycles of 94° C. for 30 seconds, 55° C. for 30seconds, 68° C. for 4 minutes; 10 cycles of 94° C. for 30 seconds, 50°C. for 30 seconds, 68° C. for 4 minutes; final extention at 68° C. for 7minutes.

A second assembly PCR was performed to combine the assembled pairs tomake Operon 6 which contained all four genes. Two end primers (SEQ IDNOS: 59 and 66) were added to the PCR mixture containing 100 ng of thepurified pair of alanine aminomutase/beta-alanine aminotransferase; 100ng of the purified pair of 3-HP dehydrogenase/alpha-alanineaminotransferase, and the mix of rTth polymerase and Pfu Turbopolymerase in a ratio of 8:1. The assembly PCR was run under thefollowing conditions: initial denaturation step 94° C. for 2 minutes; 15cycles of 94° C. for 30 seconds, 50° C. for 30 seconds, 70° C. for 5minutes; 10 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds, 70°C. for 5 minutes; final extention at 70° C. for 7 minutes.

The assembled PCR product was gel purified as described above anddigested with EcoRI and EcoRV. The sites for these restriction enzymeswere introduced to assembled PCR product with SEQ ID NO: 59 (EcoRI) andSEQ ID NO: 66 (EcoRV) primers. The digested PCR product was heated at65° C. for 30 minutes, gel purified using Qiagen Gel Extraction kit andused for ligation to pTrc99A vector digested with EcoRI and SmaI. Theligation was performed at 16° C. overnight using T4 ligase, and themixture transformed into chemically competent Tuner cells and plated onLB plates supplemented with 50 μg/ml carbenicillin. Individual colonieswere selected for plasmid DNA purification; plasmid DNA was obtainedusing Qiagen Spin Miniprep Kit. Plasmids were screened by PCR with SEQID NOS: 59 and 66 primers and analyzed by gel electrophoresis. Theresulting plasmid was named pTrcβ-ala.

Tuner(pTrcβ-ala) cells were used to determine the expression of thecloned genes and production of 3-HP from glucose, alpha-alanine andbeta-alanine. Tuner(pTrc99A) were used as a control. Cells were grown toOD₆₀₀˜0.5 in M9CA medium (Difco Laboratories) supplemented with 5 g/lglucose and 50 μg/ml carbenicillin; or 5 g/l glucose, 5 g/lalpha-alanine and 50 μg/ml carbenicillin; or 5 g/l glucose, 5 g/lbeta-alanine and 50 μg/ml carbenicillin; and induced with 100 μM IPTGand 0.5% arabinose under aerobic conditions. Production of 3-HP frombeta-alanine was detected with cells carrying pTrcβ-ala, but not in thecontrol cells by LC-MS analysis. 3-HP was observed in cell supernatantsafter 22 hours of induction.

Example 12 Production of Pantothenate from Beta-Alanine

Pantothenate can be produced from beta-alanine by a polypeptides havingalpha-ketopantoate hydroxymethyltransferase (E.C. 2.1.2.11),alpha-ketopantoate reductase (E.C. 1.1.1.169), and pantothenate synthase(E.C. 6.3.2.1) activity (FIG. 3).

Using the cloning methods described in EXAMPLES 10 and 11,alpha-ketopantoate hydroxymethyltransferase (E.C. 2.1.2.11),alpha-ketopantoate reductase (E.C. 1.1.1.169), and pantothenate synthase(E.C. 6.3.2.1) polypeptides can be isolated, sequenced, expressed, andtested. One skilled in the art will understand that similar methods canbe used to obtain the sequence of any such polypeptides from anyorganism.

Example 13 Recombinant Expression

With publicly available enzyme cDNA and amino acid sequences, and theenzymes and sequences disclosed herein, such as alanine 2,3-aminomutase,CoA transferase, beta-alanyl-CoA ammonia lyase, 3-HP—CoA dehydratase,4-aminobutyrate aminotransferase, beta-alanine-2-oxo-glutarateaminotransferase, 3-hydroxypropionate dehydrogenase,3-hydroxyisobutyrate dehydrogenase, glutamate dehydrogenase, 3-HP—CoAhydrolase, 3-hydroxyisobutryl-CoA hydrolase, poly hydroxyacid synthase,lipase, esterase, CoA hydrolase, alpha-ketopantoatehydroxymethyltransferase, alpha-ketopantoate reductase, pantothenatesynthase, pantothenate kinase, 4′-phosphopantethenoyl-1-cysteinesynthetase, 4′-phosphopantothenoylcysteine decarboxylase,ATP:4′-phosphopantetheine adenyltransferase, dephospho-CoA kinaseacetylating aldehyde:NAD(+) oxidoreductase, alcohol:NAD(+)oxidoreductase, aldehyde dehydrogenase (NAD(P)+) and alcoholdehydrogenase, as well as variants, polymorphisms, mutants, fragmentsand fusions thereof, the expression and purification of any protein,such as an enzyme, by standard laboratory techniques is enabled. Oneskilled in the art will understand that enzymes and fragments thereofcan be produced recombinantly in any cell or organism of interest, andpurified prior to use, for example prior to production of 3-HP,pantothenate and derivatives thereof.

Methods for producing recombinant proteins are well known in the art.Therefore, the scope of this disclosure includes recombinant expressionof any protein or fragment thereof, such as an enzyme. For example, seeU.S. Pat. No. 5,342,764 to Johnson et al.; U.S. Pat. No. 5,846,819 toPausch et al.; U.S. Pat. No. 5,876,969 to Fleer et al. and Sambrook etal. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,1989, Ch. 17).

Briefly, partial, full-length, or variant cDNA sequences, which encodefor a protein or peptide, can be ligated into an expression vector, suchas a bacterial expression vector. Proteins and/or peptides can beproduced by placing a promoter upstream of the cDNA sequence. Examplesof promoters include, but are not limited to lac, trp, tac, trc, majoroperator and promoter regions of phage lambda, the control region of fdcoat protein, the early and late promoters of SV40, promoters derivedfrom polyoma, adenovirus, retrovirus, baculovirus and simian virus, thepromoter for 3-phosphoglycerate kinase, the promoters of yeast acidphosphatase, the promoter of the yeast alpha-mating factors andcombinations thereof.

Vectors suitable for the production of intact native proteins includepKC30 (Shimatake and Rosenberg, 1981, Nature 292:128), pKK177-3 (Amannand Brosius, 1985, Gene 40:183) and pET-3 (Studier and Moffatt, 1986, J.Mol. Biol. 189:113). A DNA sequence can be transferred to other cloningvehicles, such as other plasmids, bacteriophages, cosmids, animalviruses and yeast artificial chromosomes (YACs) (Burke et al., 1987,Science 236:806-12). These vectors can be introduced into a variety ofhosts including somatic cells, and simple or complex organisms, such asbacteria, fungi (Timberlake and Marshall, 1989, Science 244:1313-7),invertebrates, plants (Gasser and Fraley, 1989, Science 244:1293), andmammals (Pursel et al., 1989, Science 244:1281-8), which are renderedtransgenic by the introduction of the heterologous cDNA.

For expression in mammalian cells, a cDNA sequence can be ligated toheterologous promoters, such as the simian virus SV40, promoter in thepSV2 vector (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072-6), and introduced into cells, such as monkey COS-1 cells(Gluzman, 1981, Cell 23:175-82), to achieve transient or long-termexpression. The stable integration of the chimeric gene construct may bemaintained in mammalian cells by biochemical selection, such as neomycin(Southern and Berg, 1982, J. Mol. Appl. Genet. 1:327-41) andmycophoenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072-6).

The transfer of DNA into eukaryotic, such as human or other mammaliancells, is a conventional technique. The vectors are introduced into therecipient cells as pure DNA (transfection) by, for example,precipitation with calcium phosphate (Graham and vander Eb, 1973,Virology 52:466) strontium phosphate (Brash et al., 1987, Mol. Cell.Biol. 7:2013), electroporation (Neumann et al., 1982, EMBO J. 1:841),lipofection (Felgner et al., 1987, Proc. Natl. Acad. Sci. USA 84:7413),DEAE dextran (McCuthan et al., 1968, J. Natl. Cancer Inst. 41:351),microinjection (Mueller et al., 1978, Cell 15:579), protoplast fusion(Schafner, 1980, Proc. Natl. Acad. Sci. USA 77:2163-7), or pellet guns(Klein et al., 1987, Nature 327:70). Alternatively, the cDNA can beintroduced by infection with virus vectors, for example retroviruses(Bernstein et al., 1985, Gen. Engrg. 7:235) such as adenoviruses (Ahmadet al., 1986, J. Virol. 57:267) or Herpes (Spaete et al., 1982, Cell30:295).

Example 14 Peptide Synthesis and Purification

The enzymes disclosed herein, such as alanine 2,3-aminomutase, CoAtransferase, beta-alanyl-CoA ammonia lyase, 3-HP—CoA dehydratase,4-aminobutyrate aminotransferase, beta-alanine-2-oxo-glutarateaminotransferase, 3-hydroxypropionate dehydrogenase,3-hydroxyisobutyrate dehydrogenase, glutamate dehydrogenase, 3-HP—CoAhydrolase, 3-hydroxyisobutryl-CoA hydrolase, poly hydroxyacid synthase,lipase, esterase, CoA hydrolase, alpha-ketopantoatehydroxymethyltransferase, alpha-ketopantoate reductase, pantothenatesynthase, pantothenate kinase, 4′-phosphopantethenoyl-1-cysteinesynthetase, 4′-phosphopantothenoylcysteine decarboxylase,ATP:4′-phosphopantetheine adenyltransferase, dephospho-CoA kinaseacetylating aldehyde:NAD(+) oxidoreductase, alcohol:NAD(+)oxidoreductase, aldehyde dehydrogenase (NAD(P)+) and alcoholdehydrogenase (and variants, fusions, polymorphisms, fragments, andmutants thereof) can be chemically synthesized by any of a number ofmanual or automated methods of synthesis known in the art. For example,solid phase peptide synthesis (SPPS) is carried out on a 0.25 millimole(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizerand using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,coupling with dicyclohexylcarbodiimide/hydroxybenzotriazole or2-(1H-benzo-triazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and usingp-hydroxymethylphenoxymethylpolystyrene (HMP) or Sasrin resin forcarboxyl-terminus acids or Rink amide resin for carboxyl-terminusamides.

Fmoc-derivatized amino acids are prepared from the appropriate precursoramino acids by tritylation and triphenylmethanol in trifluoroaceticacid, followed by Fmoc derivitization as described by Atherton et al.(Solid Phase Peptide Synthesis, IRL Press: Oxford, 1989).

Sasrin resin-bound peptides are cleaved using a solution of 1% TFA indichloromethane to yield the protected peptide. Where appropriate,protected peptide precursors are cyclized between the amino- andcarboxyl-termini by reaction of the amino-terminal free amine andcarboxyl-terminal free acid using diphenylphosphorylazide in nascentpeptides wherein the amino acid sidechains are protected.

HMP or Rink amide resin-bound products are routinely cleaved andprotected sidechain-containing cyclized peptides deprotected using asolution comprised of trifluoroacetic acid (TFA), optionally alsocomprising water, thioanisole, and ethanedithiol, in ratios of100:5:5:2.5, for 0.5-3 hours at RT.

Crude peptides are purified by preparative high pressure liquidchromatography (HPLC), for example using a Waters Delta-Pak C18 columnand gradient elution with 0.1% TFA in water modified with acetonitrile.After column elution, acetonitrile is evaporated from the elutedfractions, which are then lyophilized. The identity of each product soproduced and purified may be confirmed by fast atom bombardment massspectroscopy (FABMS) or electrospray mass spectroscopy (ESMS).

In view of the many possible embodiments to which the principles of ourdisclosure may be applied, it should be recognized that the illustratedembodiments are only particular examples of the disclosure and shouldnot be taken as a limitation on the scope of the disclosure. Rather, thescope of the disclosure is in accord with the following claims. Wetherefore claim as our invention all that comes within the scope andspirit of these claims.

1. An isolated nucleic acid molecule comprising a nucleic acid sequencethat encodes a polypeptide having at least 90% sequence identity to thepolypeptide sequence shown in SEQ ID NO: 21, wherein the amino acidcorresponding to position 339 of SEQ ID NO: 21 is a Gly, Gln, Thr, Asn,or His, and wherein the polypeptide has alanine 2,3-aminomutaseactivity.
 2. The isolated nucleic acid molecule of claim 1, operablylinked to a promoter sequence.
 3. A vector comprising the isolatednucleic acid molecule of claim
 1. 4. The isolated nucleic acid moleculeof claim 1, wherein the nucleic acid sequence comprises a sequencehaving at least 90% sequence identity to the nucleic acid sequence shownin SEQ ID NO:
 20. 5. The isolated nucleic acid molecule of claim 1,wherein the nucleic acid sequence comprises a sequence having at least95% sequence identity to the nucleic acid sequence shown in SEQ ID NO:20.
 6. The isolated nucleic acid molecule of claim 1, wherein thenucleic acid sequence comprises a sequence having at least 97% sequenceidentity to the nucleic acid sequence shown in SEQ ID NO:
 20. 7. Theisolated nucleic acid molecule of claim 1, wherein the nucleic acidsequence comprises a sequence having at least 98% sequence identity tothe nucleic acid sequence shown in SEQ ID NO:
 20. 8. The isolatednucleic acid molecule of claim 1, wherein the nucleic acid sequencecomprises a sequence having at least 99% sequence identity to thenucleic acid sequence shown in SEQ ID NO:
 20. 9. The isolated nucleicacid molecule of claim 1, wherein the nucleic acid comprises SEQ ID NO:20.
 10. The isolated nucleic acid molecule of claim 1, wherein thenucleic acid sequence includes one or more substitutions which resultsin one or more conservative amino acid substitutions.
 11. The isolatednucleic acid molecule of claim 1, wherein the nucleic acid sequenceincludes one or more substitutions which results in no more than 10conservative amino acid substitutions.
 12. An isolated nucleic acidmolecule comprising a nucleic acid sequence that encodes a polypeptidecomprising amino acids 15-390 of SEQ ID NO: 21, wherein the nucleic acidmolecule encodes a polypeptide having alanine 2,3-aminomutase activity.13. The isolated nucleic acid molecule of claim 12, wherein the nucleicacid sequence comprises nucleotides 307-1017 of SEQ ID NO:
 20. 14. Anisolated transformed cell comprising at least one exogenous nucleic acidmolecule, wherein the at least one exogenous nucleic acid moleculecomprises the nucleic acid molecule of claim
 1. 15. The cell of claim14, wherein the cell produces beta-alanine from alpha-alanine.
 16. Theisolated nucleic acid molecule of claim 1, wherein the amino acidcorresponding to position 339 of SEQ ID NO: 21 is a His.
 17. Theisolated nucleic acid molecule of claim 1, wherein the amino acidcorresponding to position 103 of SEQ ID NO: 21 is a Met, Lys, Arg, Glu,or Ser.
 18. The isolated nucleic acid molecule of claim 1, wherein theamino acid corresponding to position 136 of SEQ ID NO: 21 is a Val. 19.The isolated nucleic acid molecule of claim 1, wherein the amino acidcorresponding to position 103 of SEQ ID NO: 21 is a Met, Lys, Arg, Glu,or Ser, and wherein the amino acid corresponding to position 136 of SEQID NO: 21 is a Val.
 20. The isolated nucleic acid molecule of claim 1,wherein the amino acid corresponding to position 339 of SEQ ID NO: 21 isa His; the amino acid corresponding to position 103 of SEQ ID NO: 21 isa Met; and wherein the amino acid corresponding to position 136 of SEQID NO: 21 is a Val.
 21. An isolated nucleic acid molecule comprising anucleic acid sequence that encodes an polypeptide having at least 90%sequence identity to the polypeptide sequence shown in SEQ ID NO: 21,wherein the amino acid corresponding to position 103 of SEQ ID NO: 21 isa Met, Lys, Arg, Glu, or Ser, and wherein the polypeptide has alanine2,3-aminomutase activity.
 22. The isolated nucleic acid molecule ofclaim 21, operably linked to a promoter sequence.
 23. A vectorcomprising the isolated nucleic acid molecule of claim
 21. 24. Theisolated nucleic acid molecule of claim 21, wherein the nucleic acidsequence comprises a sequence having at least 90% sequence identity tothe nucleic acid sequence shown in SEQ ID NO:
 20. 25. The isolatednucleic acid molecule of claim 21, wherein the nucleic acid sequencecomprises a sequence having at least 95% sequence identity to thenucleic acid sequence shown in SEQ ID NO:
 20. 26. The isolated nucleicacid molecule of claim 21, wherein the nucleic acid sequence comprises asequence having at least 97% sequence identity to the nucleic acidsequence shown in SEQ ID NO:
 20. 27. The isolated nucleic acid moleculeof claim 21, wherein the nucleic acid sequence comprises a sequencehaving at least 98% sequence identity to the nucleic acid sequence shownin SEQ ID NO:
 20. 28. The isolated nucleic acid molecule of claim 21,wherein the nucleic acid sequence comprises a sequence having at least99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:20.
 29. The isolated nucleic acid molecule of claim 21, wherein thenucleic acid sequence includes one or more substitutions which resultsin one or more conservative amino acid substitutions.
 30. The isolatednucleic acid molecule of claim 21, wherein the nucleic acid sequenceincludes one or more substitutions which results in no more than 10conservative amino acid substitutions.
 31. An isolated transformed cellcomprising at least one exogenous nucleic acid molecule, wherein the atleast one exogenous nucleic acid molecule comprises the nucleic acidmolecule of claim
 21. 32. The cell of claim 31, wherein the cellproduces beta-alanine from alpha-alanine.
 33. The isolated nucleic acidmolecule of claim 21, wherein the amino acid corresponding to position103 of SEQ ID NO: 21 is a Met.
 34. The isolated nucleic acid molecule ofclaim 21, wherein the amino acid corresponding to position 136 of SEQ IDNO: 21 is a Val.
 35. An isolated nucleic acid molecule comprising anucleic acid sequence that encodes a polypeptide having at least 90%sequence identity to the polypeptide sequence shown in SEQ ID NO: 21,wherein the amino acid corresponding to position 136 of SEQ ID NO: 21 isa Val, and wherein the polypeptide has alanine 2,3-aminomutase activity.36. The isolated nucleic acid molecule of claim 35, operably linked to apromoter sequence.
 37. A vector comprising the isolated nucleic acidmolecule of claim
 35. 38. The isolated nucleic acid molecule of claim35, wherein the nucleic acid sequence comprises a sequence having atleast 90% sequence identity to the nucleic acid sequence shown in SEQ IDNO:
 20. 39. The isolated nucleic acid molecule of claim 35, wherein thenucleic acid sequence comprises a sequence having at least 95% sequenceidentity to the nucleic acid sequence shown in SEQ ID NO:
 20. 40. Theisolated nucleic acid molecule of claim 35, wherein the nucleic acidsequence comprises a sequence having at least 97% sequence identity tothe nucleic acid sequence shown in SEQ ID NO:
 20. 41. The isolatednucleic acid molecule of claim 35, wherein the nucleic acid sequencecomprises a sequence having at least 98% sequence identity to thenucleic acid sequence shown in SEQ ID NO:
 20. 42. The isolated nucleicacid molecule of claim 35, wherein the nucleic acid sequence comprises asequence having at least 99% sequence identity to the nucleic acidsequence shown in SEQ ID NO:
 20. 43. The isolated nucleic acid moleculeof claim 35, wherein the nucleic acid sequence includes one or moresubstitutions which results in one or more conservative amino acidsubstitutions.
 44. The isolated nucleic acid molecule of claim 35,wherein the nucleic acid sequence includes one or more substitutionswhich results in no more than 10 conservative amino acid substitutions.45. An isolated transformed cell comprising at least one exogenousnucleic acid molecule, wherein the at least one exogenous nucleic acidmolecule comprises the nucleic acid molecule of claim
 35. 46. The cellof claim 35, wherein the cell produces beta-alanine from alpha-alanine.